Design and selection of medicaments that modulate the function and activity of interleukin 13

ABSTRACT

The present invention relates generally to the field of medicaments in the form of therapeutic molecules including inflammatory modulators and their design and selection. More specifically, the present invention relates to a target site on Interleukin 13 (IL-13) by which a GAG molecule or polyanionic glycoconjugate or anionic polysaccharide modulates IL-13 activity or function, said target site selected from the list consisting of amino acids located in the AB loops and/or helix D of human IL-13 or its homolog or derivative, and the use of said IL-13 target site to design a medicament for modulating physiological processes. Therapeutic and prophylactic compositions comprising the designed medicaments are also contemplated.

FILING DATA

This application is a divisional of application Ser. No. 12/809,968,filed Sep. 22, 2010, which is a U.S. National Phase of InternationalApplication No. PCT/AU2008/001871, filed Dec. 19, 2008, which claims thebenefit of Australian Application No. 2007907059, filed Dec. 21, 2007,all of which are hereby expressly incorporated by reference in theirentireties.

FIELD

The present invention relates generally to the field of medicaments inthe form of therapeutic molecules including inflammatory modulators andtheir design and selection. Therapeutic and prophylactic compositionscomprising the medicaments are also contemplated.

BACKGROUND

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

The design and selection of therapeutic molecules having a high degreeof target specificity is a major goal of pharmaco-mimetic research. Withthe rapid development of complex computational algorithms, in silicoscreening is now a reasonable approach to rational drug design. Thedifficulty, however, is the identification of not only target moleculesbut conformationally active pockets, clefts and sites on thesemolecules.

The identification of therapeutic molecules useful in inflammation isparticularly important.

Inflammation is a complex multifactorial process, which includes themigration of neutrophils and monocytes from blood into tissue atinflammatory sites. This migration involves a series of sequential stepsproceeding from tethering on endothelium under shear conditions inpostcapillary venules (Smith, Microcirculation 7:385-394, 2000). Thetethering event depends on adhesion molecules in the selectin family,E-selectin and P-selectin on the endothelium and L-selectin on theneutrophil as well as their respective ligands expressed on both celltypes (Burns et al, Physiol Rev 83:309-336, 2003). The adhesion stepprimarily involves the interaction of integrins (αLβ2, αMβ2, α4β7, α4β1)with adhesion molecules of the Ig-superfamily (ICAM-1, ICAM-2, MadCAMand VCAM) (Fabbri et al, Inflamm Res 48:239-246, 1999). Whereas, thetransendothelial cell migration step involves molecules expressed at thejunctions between adjacent endothelial cells.

Chronic inflammatory diseases affecting the lung such as bronchialasthma, chronic obstructive pulmonary disease (COPD) and allergicrhinitis are particularly problematic which cause high levels ofmorbidity and mortality. Asthma is particularly prevalent and is oftentriggered by exposure to environmental stimuli such as allergens,pharmacological agents, infectious agents, airborne pollutants andirritants.

One cytokine associated with inflammatory lung conditions isinterleukin-13 (IL-13). IL-13 is a 17 kDa glycoprotein which has beencloned from activated T-cells (Zurawski and dVries, Immunol Today15:19-26, 1994). It is a member of the cytokine family characterized bya tertiary structure of four α-helical bundles. The helices are definedas helix A through D. The turns in the helices are referred to as loops,i.e. AB, BC and CD loops. Other members of this family include IL-2,IL-3, IL-4, IL-5 and GM-CSF. It was originally described as a Tcell-derived cytokine that inhibits inflammatory cytokine production,however numerous other functions are also attributable to IL-13. Theseinclude the regulation of gastrointestinal parasite expulsion, airwayhyperresponsiveness (AHR), allergic inflammation, atopic dermatitis,chronic obstructive pulmonary disease (COPD), tissue eosinophilia,mastocytosis, IgE antibody production, goblet cell hyperplasia, tumorcell growth, intracellular parasitism, tissue remodeling and fibrosis(Wynn, Annu. Rev. Immunol. 21:425-56, 2003). The gene encoding humanIL-13 is located on chromosome 5Q31, in the same 3000 kb cluster ofgenes that encodes IL-3, IL-4, IL-5, IL-9 and GM-CSF. The IL-13 gene is12 kb upstream from the gene encoding IL-4 and lies in the sameorientation, indicating that these genes arose by gene duplicationduring evolution (de Waal Malefyt and de Vries, The Cytokine Handbook,3rd Edn. A. W. Thomson, Editor: 427-442, 1998). The IL-13 protein hasonly 25% homology with IL-4 but it does display some similarities offunction due to the sharing of a receptor complex with IL-4.

The IL-13 receptor complex includes the IL-4 receptor (IL-4Rα) chain andtwo other IL-13 binding proteins, IL-13Rα1 and IL-13Rα2. Both IL-13Rα1and IL-13 Rα2 bind IL-13 but only IL-13Rα1 interacts with IL-4 despiteIL-13Rα2 sharing a 37% homology with IL-13Rα1 (Andrews et al, J. AllergyClin. Immunol 120:91-97, 2007). IL-13Rα1 by itself binds IL-13 withlow-moderate affinity (2-10 nM), but in the presence of the IL-4Rαchain, it binds IL-13 with high affinity (kd ˜300-400 μM). In contrast,IL-13 Rα2 binds IL-13 with high affinity (0.5-1.2 nM) affinity but itappears not to contribute to IL-13 signaling. It has been suggested thatit acts as a decoy receptor. Thus, the heterodimeric complex formed bythe IL-13Rα1 and IL-4Rα chains constitutes the functional IL-13 receptor(Wills-Karp, Immunol. Rev. 202:175-190, 2004). There is a sequentialbinding sequence for this receptor complex where IL-13 first bindsIL-13Rα1 and then recruits IL-4Rα to form a high affinity binding site(Andrews et al, J. Immunol. 176:7456-7461, 2006). Heterodimerization ofIL-13R causes activation of Janus kinases, TYK2 and JAK1, constitutivelyassociated with IL-13Rα1 and IL-4Rα, respectively, followed byactivation of the signal transducer and activator of transcription 6(STAT6).

The IL-13R (IL-4Rα/IL-13Rα1) is expressed on hemopoietic andnon-hemopoietic cells including B cells, monocytes/macrophages,dendritic cells, eosinophils, basophils, fibroblasts, endothelial cells,airway epithelial cells and airway smooth muscle cells. IL-13 Rα2 hasbeen found on airway smooth muscle cells and airway epithelial cells.IL-13 Rα2 has been described as a decoy receptor and a fusion proteinconsisting of IL-13 Rα2 and the Fc portion of immunoglobulin is used asan inhibitor of IL-13 in vitro and in vivo. IL-4 can also use IL-13R(IL-4Rα/IL-13 Rα1) as well as another receptor consisting of IL-4Rα andthe common γ-chain of the IL-2 receptor. This overlap of receptorsaccounts for many of the functional similarities of IL-4 and IL-13(Andrews et al, 2006 supra).

The sites on the IL-13 molecule recognized by each of the receptorchains differ. For example molecular modeling has suggested that theD-helix of IL-13 interacts with IL-13Rα1 whereas parts of the A andC-helices of IL-13 interact with the IL-4Rα chain of the IL-13R (Oshimaand Puri, J. Biol. Chem. 276:15195-15191, 2001; Zuegg et al, Immunol.Cell Biol. 79:332-339, 2001). Mutations of glutamic acids at positions12 and 15 in helix A and arginine and serine at positions 65 and 68respectively in the C helix were found to be important for biologicalsignaling through the IL-4Rα chain since their specific mutationresulted in loss and/or gain of function (Thompson and Debinski, J.Biol. Chem. 274:29944-29950, 1999). Indeed, mutation E12K produced apowerful antagonist that inhibits the activities of human IL-13 (Oshimaand Puri, 2001 supra). Whereas amino acids in the D-helix have beendescribed as important for binding to IL-13Rα1 and/or IL-13Rα2, theseare H102, K104, K105, R108, E109 and R111 (Arima et al, J. Biol. Chem.280:24915-24922, 2005; Madhankumar et al, J. Biol. Chem.277:43194-43205, 2002). These data are supported by molecular modelingstudies and the crystal structures of the signaling complex ofIL-4Rα/IL-13/IL-13Rα1. Analyses of the crystal structure further suggestK104 and R108 on IL-13 are critically important for the interactionswith IL-13Rα1 domain 3 (LaPorte et al, Cell 132:259-272, 2008). A stripeof amino acids on the A and D helices demarcates a hydrophobic canyonlined by the alkyl moieties of these amino acids. These side chains formclefts into which the receptors insert to contact the main chains of thecytokine A and D helices and of particular importance are the sidechains of amino acids R108 and K104 on the D helix. Whereas it appearsthat R111 is important for binding to the soluble receptor IL-13Rα2(Andrews et al, J. Allergy Clin. Immunol. 120:91-97, 2007). Domain 1 ofIL-13Rα1 interacts with a hydrophobic saucer-shaped patch formed by thealkyl side chains of M33, D87, K89, T35 (LaPorte et al, 2008 supra).

The ability to modify IL-13 function would aid in the development ofmedicaments useful in the treatment of inflammatory conditions of thelung including asthma, anaphylaxis, emphysema and COPD, and otherdiseases to which IL-13 contributes including atopic dermatitis,fibrosis and various cancers, for example B chronic lymphocytic leukemia(B-CLL), Hodgkin's disease, where tumor growth/protection from apoptosisis promoted by IL-13, and other cancers in which IL-13 appears toantagonize tumor immunosurveillance (Wynn, 2003 supra). The link betweenIL-13 and fibrosis suggests that IL-13 antagonists may also be effectivein a variety of situations where chronic exposure to IL-13 triggersexcessive healing, tissue remodeling, or the formation of destructivetissue pathology in situations like idiopathic pulmonary fibrosis,chronic graft rejection, bleomycin-induced pulmonary fibrosis,progressive systemic sclerosis, radiation-induced pulmonary fibrosis,hepatic fibrosis and acute respiratory distress syndrome (ARDS).

Particular glycosaminoglycan (GAG) sequences can bind specifically andmake unique interactions with a number of biomolecules includingchemokines, growth factors, cytokines, proteins of the coagulationcascade (e.g. anti-thrombin III (AT-III)-pentasaccharide complex) andadhesion molecules. Very few GAG fragments, however, have been developedfor therapeutic use, mostly because the synthesis of saccharide blocksis chemically challenging. The antithrombin (AT)-binding pentasaccharideArixtra (Registered trade mark) [Sanofi] has been approved for use inthromboprophylaxis following orthopedic surgery and the GAG mimeticPI-88 (Progen) has progressed to clinical trials as an anti-cancertreatment. Other polyanionic polysaccharides (for example pentosanpolysulfate) can also bind biomolecules including chemokines, growthfactors, cytokines, proteins of the coagulation cascade (e.g.anti-thrombin III (AT-III)-pentasaccharide complex) and adhesionmolecules in a manner resembling that of GAGs or GAG sequences.

There is a need to delineate the nature of the interactions of IL-13with GAGs. This will enable development of small molecule selectivemodulators of GAG-IL-13 interactions. This includes blocking IL-13signaling via its receptor (IL-13R) to thereby treat disease conditionsthat arise because of this signaling event.

SUMMARY

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

A structural model of the extracellular domains of IL-13, the locationof sulfate binding regions and the modeling of the binding of variousheparin and GAG fragments to IL-13 are provided herein. This has lead tothe identification of the interactions and affinity of heparin and GAGfragments of various sizes to a particular binding site on IL-13. Thisin turn enables the determination of a molecular target for the designand selection of GAG-like molecules that are smaller than naturallyoccurring GAGs. It is proposed that these molecules that are GAG-like innature but are smaller than naturally occurring GAGs (GAGmimetics/polyanionic glycoconjugates) have potential as therapeutic andprophylactic agents to modulate IL-13 activity and functions becausethey bind to IL-13 at a site that overlaps with the IL-13Rα1 and/or theIL-13Rα2 binding sites, thereby blocking IL-13 from interacting withthese molecules and as a consequence blocking the formation of theIL-4Rα/IL-13 Rα1 complex which constitutes the functional IL-13receptor. Hence, GAG mimetics/polyanionic glycoconjugates have a role astherapeutic and prophylactic agents in a range of disease processes suchas inflammation, fibrosis, chronic graft rejection, cancer and stem celldifferentiation and proliferation. In particular the allergicinflammatory diseases asthma, allergic rhinitis and atopic dermatitis oreczema and other inflammatory respiratory diseases like COPD and ARDSare likely to be positively affected by these molecules that areGAG-like in nature but are smaller than naturally occurring GAGs.

IL-13 comprises four helical bundles. Each helix is referred to as helixA, helix B, helix C and helix D, respectively. The turns in the helicesare referred to as loops, hence AB, BC and CD loops. The amino acidnumbering system used in this specification is that given in the humanIL-13 monomer structure; 1ijz.pdb.

Accordingly, one aspect of the present invention is directed to a targetsite on IL-13 at which a GAG molecule or a polyanionic glycoconjugatemodulates IL-13 activity or function, the target site comprising aminoacids located on a region selected from the list consisting of the ABloop, helix D and a combination of the AB loop and helix D. TheGAG/polyanionic glycoconjugate binding site overlaps the binding site onthe D-helix of IL-13 for receptors, IL-13Rα1 and/or IL-13Rα2, therebyblocking IL-13 from interacting with these molecules and as aconsequence blocking the formation of the IL-4Rα/IL-13 Rα1 complex whichconstitutes the functional IL-13 receptor.

A target site for the design and selection of IL-13 activity or functionmodifiers is further provided comprising a conformation of amino acidresidues within a region selected from the list consisting of the ABloop, helix D and a combination of the AB loop and helix D.

Another aspect of the present invention provides a GAG or a polyanionicglycoconjugate binding site on IL-13 useful as a target site for thedesign and selection of IL-13 activity or function modifiers, the GAGbinding site selected from the listing consisting of:

(i) a conformation of amino acid residues in the AB loop comprisingamino acid residues Q22, Q24 and K25;(ii) a conformation of amino acid residues on helix D selected fromamino acid residues K97, D98, H102, K104, K105, R108, E109 and R111; and(iii) a conformation of amino acid residues selected from amino acidresidues Q22, Q24 and K25 in the AB loop and K97, D98, H102, K104, K105,R108, E109 and R111 in helix D.

These amino acids are in human IL-13 but the present invention extendsto derivative IL-13 molecules including splice variants and polymorphicvariants and the equivalent location in non-human IL-13 homologs.

Another aspect is directed to a GAG or a polyanionic glycoconjugatebinding site on IL-13 useful as a target site for the design andselection of IL-13 activity or function modifiers, the GAG/polyanionicglycoconjugate binding site comprising amino acids Q22, Q24 and K25 inthe AB loop and K97, D98, H102, K104, K105, R108, E109 and R111 in helixD.

A further aspect of the present invention is directed to an isolatedagent which modifies IL-13 activity or function the agent capable ofinteracting with or antagonizing or agonizing binding of a GAG or apolyanionic glycoconjugate to a site on IL-13 selected from the listconsisting of amino acid residues in the AB loop, amino acid residues inhelix D and amino acid residues in the AB loop and helix D.

More particularly, the present invention provides an isolated agent,which interacts with or antagonizes or agonizes binding of a GAG or apolyanionic glycoconjugate to a site on IL-13 selected from the listconsisting of:

(i) a conformation of amino acid residues in the AB loop comprisingamino acid residues Q22, Q24 and K25 in human IL-13 or its equivalent innon-human IL-13;(ii) a conformation of amino acid residues in helix D selected fromamino acid residues K97, D98, H102, K104, K105, R108, E109 and R111 ofhuman IL-13 or its equivalent in non-human IL-13; and(iii) a conformation of amino acid residues selected from amino acidresidues Q22, Q24 and K25 in the AB loop and K97, D98, H102, K104, K105,R108, E109 and R111 in helix D of human IL-13 or its equivalent innon-human IL-13.

Reference to “IL-13” includes mutants, derivatives, splice variants,polymorphism variants and the like. It particularly includes thenaturally occurring human IL-13 variant IL-13R111Q, where an arginine atposition 111 has been substituted with a glutamine. This variant isfound in approximately 20% of the Caucasian population and a number ofstudies have shown strong associations between this IL-13 polymorphismand atopy and atopic diseases such as asthma, atopic dermatitis andallergic rhinitis (Andrews, 2007 supra).

The present invention also contemplates the use of a GAG or polyanionicglycoconjugate binding site on IL-13 in the design of a medicament formodulating physiological processes in a subject such as inflammatoryprocesses.

Another aspect of the present invention is that heparin, a heparinoligosaccharide or a fraction or derivative of heparin acts as anantagonist of IL-13 because it binds to IL-13 at a site selected fromthe list consisting of:

(i) amino acid residues Q22, Q24 and K25 in the AB loop;(ii) amino acid residues K97, D98, 11102, K104, K105, R108, E109 andR111 on helix D; and(iii) amino acid residues Q22, Q24 and K25 in the AB loop and K97, D98,H102, K104, K105, R108, E109 and R111 on helix D.

Another aspect of the present invention is that a fraction or derivativeof the sulfated xylan, pentosan polysulfate (PPS) acts as an antagonistof IL-13 because it binds to IL-13 at a site selected from the listconsisting of:

(i) amino acid residues Q22, Q24 and K25 in the AB loop;(ii) amino acid residues K97, D98, H102, K104, K105, R108, E109 and R111on helix D; and(iii) amino acid residues Q22, Q24 and K25 in the AB loop and K97, D98,H102, K104, K105, R108, E109 and R111 on helix D.

The present invention further contemplates sulfate binding motifs on theAB loop and/or helix D selected from the list consisting of:

(i) amino acid residues Q22, Q24 and K25 on the AB loop in human IL-13or its equivalent in non-human IL-13;(ii) amino acid residues K97, D98, H102, K104, K105, R108, E109 and R111in helix D of human IL-13 or its equivalent in non-human IL-13; and(iii) an amino acid residues Q22, Q24 and K25 in the AB loop and aminoacid residues K97, D98, H102, K104, K105, R108, E109 and R111 on helix Dof human IL-13 or its equivalent in non-human IL-13.

A further aspect provides the use of sulfate binding motifs in the ABloop and/or on helix D of IL-13 selected from the list consisting of:

(i) amino acid residues Q22, Q24 and K25 in the AB loop in human IL-13or its equivalent in non-human IL-13;(ii) amino acid residues K97, D98, H102, K104, K105, R108, E109 and R111on helix D of human IL-13 or its equivalent in non-human IL-13; and(iii) amino acid residues Q22, Q24 and K25 in the AB loop and K97, D98,H102, K104, K105, R108, E109 and R111 on helix D of human IL-13 or itsequivalent in non-human IL-13;in the manufacture of a medicament for modulating physiologicalprocesses including inflammatory processes.

The present invention also contemplates a method of treatment orprophylaxis in a subject the method comprising administering to asubject an IL-13 activity or function modifier which binds or interactswith a GAG or polyanionic glycoconjugate binding site on IL-13 selectedfrom the list consisting of:

(i) amino acid residues Q22, Q24 and K25 in the AB loop;(ii) amino acid residues K97, D98, H102, K104, K105, R108, E109 and R111on helix D; and(iii) amino acid residues Q22, Q24 and K25 in the AB loop and K97, D98,H102, K104, K105, R108, E109 and R111 on helix D.

Methods of design and selection of IL-13 activity and function modifiersincluding in silico screening and other computer-based methods arefurther contemplated herein.

Single and three letter abbreviations for amino acid residues usedherein are defined in Table 1.

TABLE 1 Amino Acid Abbreviations Amino Acid Three-letter AbbreviationOne-letter Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Asparticacid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E GlycineGly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys KMethionine Met M Phenylalamine Phe F Proline Pro P Serine Ser SThreonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

BRIEF DESCRIPTION OF THE FIGURES

Some figures contain color representations or entities. Colorphotographs are available from the Patentee upon request or from anappropriate Patent Office. A fee may be imposed if obtained from aPatent Office.

FIGS. 1A and B are a graphical representations showing (A) the bindingof human IL-13 to heparin immobilized on a BIAcore chip as determined bysurface Plasmon resonance. IL-13 at the concentrations indicated wasinjected over heparin immobilized onto streptavidin sensor chips. Thebinding data were monitored during both the association and dissociationphases. (B) IL-13 binding to either the heparin coupled flow cell (fc2)or the blank flow cell (fc1). Plotted is the data for flow cell 2 minusthat of flow cell 1 (fc2-1).

FIGS. 2A and B are graphical representations showing the ability ofvarious anionic polysaccharides to block the cell proliferation activityof IL-13. (A) the effect of heparin or sucrose octasulfate (both at 10μg/ml) on the IL-13 mediated cell proliferation of TF1.8 cells; atitration of IL-13 is shown. (B) is a graphical representation showingthe dose-response curves of pentosan (dashed line) and heparin (fullline) for IL-13 dependent proliferation of TF1.8 cells at 25 ng/ml ofIL-13.

FIGS. 3A and B are graphical representations showing the ability ofheparin and heparan sulfate fragments to bind to IL-13 and to inhibitIL-13 activity is dependent upon fragment size. (A) Graphicalrepresentation showing the bidnign of IL-13R111 to immobilized heparinon a BIAcore chip as determined by surface Plasmon resonance. IL-13 (75nM) was injected alone or following preincubation with heparin (100 nM),heparan sulfate (100 nM), heparin DP10 (100 nM) and heparin DP6 (100nM). (B) Graphical representation of TF1 cell proliferation in thepresence of various sizes of heparin (hep) and heparan sulfate (HS)fragments. The saccharides were tested at 1 μg/ml and 2.5 μg/ml.

FIG. 4 is a graphical representation of showing the ability ofsulfated-xylans of sizes DP2, DP4, DP5, DP7 and DP8 to block the IL-13dependent proliferation of TF-1 cells. The different sizedsulfated-xylans are used at 10 and 2.5 μg/ml and the IL-13 is at 2.5ng/ml. The mean % inhibition+/−standard deviation of 3 replicates isshown.

FIG. 5 is a representation showing the location of amino acids thataffect human IL-13 WTbinding to GAGs or polyanioinc glycoconjugates. (A)Molecular modeling of the interaction of a rigid heparinendecasaccharide with the key IL-13 amino acids being represented assticks. Ten different docking orientations of the heparin fragment areshown. The heparin chains are displayed as a line colored by elementwhereas the protein is displayed as a ribbon.

FIG. 6 is a graphical representation of the binding of various IL-13mutants to immobilized heparin. Heparin was immobilized on a BIAcoresensor surface and the various IL-13 molecules (75 ngm in BisTris pH6.00, 0.15M NaCl 0.005% Tween 20) were passed over the surface in thefluid phase The blank is a buffer alone passed over a surface withoutcoupled heparin. (A) The binding of wild type IL-13 with arginine in the111 position (R111 WT) is compared to IL-13 mutants where a glutamine isat the 111 position, to represent the naturally occurring polymorphismof 20% of the Caucasian population, and a second mutation on the Q111background where the following amino acids have been converted toalanine: K25, K97, K104, K105 and R108. (B) The binding to immobilizedheparin of wild type IL-13 with arginine in the 111 position (IL-13R111)is compared to wild type IL-13 where lysine at position 97 has beenmutated to alanine (IL-13R111 K97A); IL-13 with glutamine at position111 (IL-13 Q111), and IL-13 with glutamine at position 111 and thelysine at position 97 has been mutated to alanine (IL-13Q111 K97A).

FIG. 7 is a graphical representation of the biological activity of thetwo polymorphic forms of human IL-13, IL-13 with arginine at position111 and IL-13 with glutamine at position 111. The biological activity ismeasured as the IL-13 induced proliferation of TF-1 cells. (A) The IL-13dependent proliferation of TF-1 cells is identical regardless of whatpolymorphic form is assayed. (B) Heparin is very effective at inhibitingthe biological activity of both forms of IL-13.

FIGS. 8A and B are diagrammatic representations of space filed models ofIl-13. Heparin fragments bind to both naturally occurring forms of IL-13at the same location and in both cases heparin binding partially masksthe site on IL-3 that is recognized by domain 3 of IL-13Rα1. (A) Spacefilled model of human IL-13Q111 binding a heparin llmer. (B) Spacefilled model of human IL-13R111 (wild-type) binding a heparin 11 mer.The amino acids involved in binding the domains 1 and 3 of IL-13Rα1 areshown. Amino acids in the D helix are shown in various shades of greenwhereas those in the A helix are in various shades of red and orange.The heparin fragments are shown as lines colored by element. Tendifferent docking orientations are shown.

DETAILED DESCRIPTION

As used in the subject specification, the singular forms “a”, “an” and“the” include plural aspects unless the context clearly dictatesotherwise. Thus, for example, reference to “a target site” includesreference to a single target site or more than one target site;reference to “an amino acid” includes a single amino acid, as well astwo or more active amino acids; reference to “the invention” includesreference to single or multiple aspects of an invention; and so forth.

The present invention provides target sites on IL-13 for the design andselection of specific antagonists and agonists of IL-13 function andactivity. Such antagonists and agonists are proposed herein to be usefulin modulating physiological processes including inflammation, cancer andstem cell differentiation and/or proliferation. All such conditions areconveniently encompassed herein by use of the term “physiologicalprocesses”. Antagonists of IL-13 activity or function are particularlyencompassed for use in the treatment of inflammation and in particularallergic inflammation.

Reference to “inflammation” or “inflammatory processes” include but arenot limited to those diseases and disorders which result in a responseof redness, swelling, pain, and a feeling of heat in certain areas thatis meant to protect tissues affected by injury or disease. Inflammatorydiseases which can be treated using the methods of the presentinvention, include, without being limited to, acne, allergic rhinitis,angina, arthritis, aspiration pneumonia, empyema, gastroenteritis,inflammation, intestinal flu, NEC, necrotizing enterocolitis, pelvicinflammatory disease, pharyngitis, PID, pleurisy, raw throat, redness,rubor, sore throat, stomach flu and urinary tract infections, chronicinflammatory demyelinating polyneuropathy, chronic inflammatorydemyelinating polyradiculoneuropathy, chronic obstructive pulmonarydisease (COPD), emphysema, asthma, acute respiratory distress syndrome(ARDS), Crohn's disease, ulcerative colitis, inflammatory bowel disease,systemic lupus erythematosus, rheumatoid arthritis, Alzheimer's disease,type I diabetes, gingivitis, eczema (atoptic dermatitis), psoriaticarthritis, tendinitis and multiple sclerosis. Allergic rhinitis, asthma,COPD, ARDSand atopic dermatitis are particular conditions contemplatedherein.

In accordance with the present invention, amino acid residues in the ABloop, helix D or a combination of the AB loop and helix D of IL-13define structural determinants for binding of GAG molecules orpolyanionic glycoconjugates. This enables the development of GAG-likemolecules and the design and selection of GAG-like molecules that aresmaller than naturally occurring GAGs, which bind to IL-13 and modifyits activity or function. Such molecules in turn are proposed to beuseful drug candidates to modulate physiological events includinginflammatory processes like asthma, anaphylaxis, emphysema, COPD atopicdermatitis and other diseases to which IL-13 contributes includingfibrosis and various cancers, for example B chronic lymphocytic leukemia(B-CLL), Hodgkin's disease, where tumor growth/protection from apoptosisis promoted by IL-13, and other cancers in which IL-13 appears toantagonize tumor immunosurveillance. The link between IL-13 and fibrosissuggests that IL-13 antagonists may also be effective in a variety ofsituations where chronic exposure to IL-13 triggers excessive healing,tissue remodeling, or the formation of destructive tissue pathology insituations like idiopathic pulmonary fibrosis, ARDS, chronic graftrejection, bleomycin-induced pulmonary fibrosis, progressive systemicsclerosis, radiation-induced pulmonary fibrosis and hepatic fibrosis andto inhibit the growth and development of certain cancers.

Various approaches have been used to identify heparin/GAG binding siteson the surface of proteins on the basis of amino acid composition(Caldwell et al, Int J Biochem Cell Biol 28(2):203-216, 1996; Fromm etal, Arch Biochem Biphys 343(1):92-100, 1997), secondary structure(Hileman et al, Bioessays 20(2):156-167, 1998) spatial distribution ofthe basic amino acids (Margalit et al, J Biol Chem 268(26):156-167,1993) and the surface properties of proteins (Forster and Mulloy,Biochem Soc Trans 34(3):431-434, 2006). While consensus sequences suchas XBBXBX and XBBBXXBX (where B is a basic residue and X can be anyresidue) have been suggested for heparin binding (Cardin and Weintraub,Arteriosclerosis 9(421-32, 1989), they are neither necessary norsufficient to define a GAG binding site. GAG binding sites generallyconsist of a cluster of basic residues on the protein surface, but notnecessarily in a continuous sequence. Moreover, even in a family ofstructurally related proteins, like the cytokine family characterized bya tertiary structure of four α-helical bundles, GAG binding sites arenot necessarily located on the same helices. Thus, the GAG binding siteon IL-4 involves the C-helix, the GAG binding site on IL-5 involves theC-helix and the β-pleated sheet consisting of the AB-loop of one monomerand the CD-loop of the other monomer (International Patent PublicationNo. WO 2005/100374 A1). This means the GAG binding site on a proteincannot be determined or predicted from the linear amino acid sequencenor from the tertiary structure of a protein.

Site directed mutagenesis involves the mutation of one or nucleotideswithin the DNA coding sequence of a protein, such that the expressedprotein is altered in at least one amino acid. The mutant can then bescreened in the heparin-binding assays described in this document forthe effect on heparin binding. Mutants that affect heparin binding canbe mapped on two or three dimensional protein models to ascertain theirposition in relation to each other, and to the protein as a whole. Thistechnique can be used on any GAG-binding protein to gather informationon any GAG-binding site on that protein [Tsiang, et al, J. Biol. Chem.270: 16854-16863, 1995].

Accordingly, one aspect of the present invention is directed to a targetsite on IL-13 at which a GAG molecule or polyanionic glycoconjugatemodulates IL-13 activity or function, the target site comprising aminoacids located on a region selected from the list consisting of the ABloop and helix D.

Reference to a “target site” includes a conformational binding regioncomprising amino acid residues within IL-13 to which a GAG molecule or apolyanionic glycoconjugate interacts. It is a target site for a GAGmolecule or for the design or selection of molecules which mimic orantagonize or promote GAG binding to IL-13 thereby increasing ordecreasing IL-13 activity or function. These molecules are GAG-like butare smaller than naturally occurring GAGs. Hence, the target site is forthe design and selection of IL-13 activity or function modifiers.

Accordingly, a target site for the design and selection of IL-13activity or function modifiers is provided comprising a conformation ofamino acid residues within the AB loop and/or helix D of IL-13.

The target sites in the AB loop and helix D can also be defined in termsof sulfate binding motifs. Hence, the present invention contemplatessulfate binding motifs in the AB loop and helix D of IL-13 selected fromthe list consisting of

(i) amino acid residue Q22, Q24 and K25 of the AB loop in human IL-13 orits equivalent in non-human IL-13;(ii) amino acid residues K97, D98, H102, K104, K105, R108, E109 and R111in helix D of human IL-13 or its equivalent in non-human IL-13; and(iii) amino acid residue Q22, Q24 and K25 of the AB loop and amino acidresidues K97, D98, H102, K104, K105, R108, E109 and R111 in helix D ofhuman IL-13 or its equivalent in non-human IL-13.

As indicated above, the modifiers may be a GAG, a GAG compositemolecule, a polyanionic glycoconjugate or any GAG-like molecule smallerthan naturally occurring GAGs that may or may not comprise saccharidematerial.

As used herein the terms “GAG-composite” structures or molecules or“composite” structures or molecules are used interchangeably. In oneembodiment, a GAG-composite structure comprises a saccharide structurethat binds a target protein. In a particular aspect, the saccharidestructure comprises two or more high charged (e.g. sulfated orphosphorylated) disaccharides or trisaccharides or tetrasaccharides orpentasaccharides or hexasaccharides or heptasaccharides oroctasaccharides or any combination of these saccharides separated by alinker or linkers. The linker is not necessarily based on a GAG-likebackbone. Rather, a linker such as an alkyl chain or a polyol structure,or polyethylene glycol is preferable. Further, it is not necessary forthe highly charged saccharides to be based on GAG structures. Othersugars, such as a mannan, or chitosan, or xylan or dextran for example,may be used as the scaffold upon which to display the charged groups.

The term “modifiers” includes antagonists and agonists of IL-13 activityor function.

It is proposed herein that amino acid conformational sites on the ABloop and/or helix D are GAG binding sites.

Reference to a “conformation of amino acid residues” includes a pocket,cleft, face or region to which a GAG molecule or a polyanionicglycoconjugate binds on IL-13. It is proposed herein that in the AB loopamino acid residue Q22, Q24 and K25 and/or on helix D, amino acidresidues K97, D98, H102, K104, K105, R108, E109 and R111 is/are involvedin a GAG binding site of human IL-13 or its derivatives or polymorphicversions of human IL-13 in particular the IL-13 variant in whicharginine at position 111 has been replaced by a glutamine and or theequivalent location in a non-human IL-13 molecule.

Reference herein to a “conformation” includes the binding pocket, cleft,face or region comprising sequential or non-sequential amino acidresidues.

Hence, another aspect is directed to a GAG/polyanionic glycoconjugatebinding site on IL-13 useful as a target site for the design andselection of IL-13 activity or function modifiers the GAG binding siteselected from the listing consisting of:

(i) a conformation of amino acid residues in the AB loop comprisingresidue Q22, Q24 and K25;(ii) a conformation of amino acid residues on helix D selected fromamino acid residues K97, D98, H102, K104, K105, R108, E109 and R111;and;(iii) a conformation of amino acid residues selected from amino acidresidue Q22, Q24 and K25 in the AB loop and K97, D98, H102, K104, K105,R108, E109 and R111 in helix D.

The amino acid residue numbers given above are for human IL-13 using asingle letter abbreviation for amino acid residues. The abbreviationsfor amino acid residues are defined in Table 1. However, the presentinvention extends to human homologs such as derivatives and splicevariants an and in particular the IL-13 variant where arginine atposition 111 is replaced with a glutamine as well as non-human homologssuch as but not limited to, from mouse, rat, rabbit, guinea pig, pig(including domestic pig and wild boar), horse, sheep, cat, dog, canalidand cow IL-13 homologs. Natural variations between various IL-13homologs may occur and hence amino acid residue numbers may change.

Hence, another aspect is directed to a GAG or a polyanionicglycoconjugate binding site on IL-13 useful as a target site for thedesign and selection of IL-13 activity or function modifiers the GAGbinding site selected from the listing consisting of:

(i) a conformation of amino acid residues comprising amino acid residuesQ22, Q24 and K25 in the AB loop of human IL-13 or its equivalent in anon-human IL-13;(ii) a conformation of amino acid residues on helix D selected fromamino acid residues K97, D98, H102, K104, K105, R108, E109 and R111 onhuman IL-13 or the equivalent in a non-human IL-13; and(iii) a conformation of amino acid residue selected from amino acidresidues Q22, Q24 and 1(25 in the AB loop of human IL-13 and K97, D98,H102, K104, K105, R108, E109 and R111 or helix D of human IL-13 or theequivalent in a non-human IL-13.

As indicated above, reference to “human IL-13” or any non-human IL-13includes any derivatives or splice variants thereof.

It is clear that the IL-13 modifiers will have human and veterinaryapplications as well as animal husbandry applications such as in thehorse, camel and dog racing industries and applications in wild animalcontrol and protection. All such applications are encompassed by thepresent invention.

Particular amino acid residues forming the GAG binding site in the ABloop include Q22, Q24 and K25 and on helix D, K97, D98, H102, K104,K105, R108, E109 and R111 of human IL-13.

Accordingly, another aspect of the present invention contemplates a GAGor an polyanionic glycoconjugate binding site on IL-13 useful as atarget for the design and selection of IL-13 activity or functionmodifiers; the GAG or polyanionic glycoconjugate binding site selectedfrom the list consisting of:

(i) amino acid residues Q22, Q24 and K25 in the AB loop of human IL-13or its equivalent in non-human IL-13 or a functional portion or regionthereof;(ii) amino acid residues K97, D98, H102, K104, K105, R108, E109 and R111on helix D of human IL-13 or its equivalent in non-human IL-13 or afunctional portion or region thereof; and(iii) amino acid residues Q22, Q24 and K25 in the AB loop and K97, D98,H102, K104, K105, R108, E109 and R111 on helix D of human IL-13 or itsequivalent in non-human IL-13.

Reference to a “functional portion or region” in relation to the GAG orpolyanionic glycoconjugate binding site means that through natural orartificial selection or mutagenesis, one or more of the defined aminoacid residues may be removed or modified without removing GAG orpolyanionic glycoconjugate binding ability. Hence, the present inventionis not necessarily limited to each group of amino acid residues in theirentirety.

Another aspect of the present invention is directed to a GAG orpolyanionic glycoconjugate binding site on IL-13 useful as a target forthe design and selection of IL-13 activity or function modifiers the GAGbinding site comprising Q22, Q24 and K25 in the AB loop and K97, D98,H102, K104, K105, R108, E109 and R111 on helix D human IL-13 or theequivalent in non-human IL-13.

The present invention further provides synthetic peptides comprisingthree or more amino acid residues comprising Q22, Q24 and K25 in the ABloop and/or comprising one or more of K97, D98, H102, K104, K105, R108,E109 and R111 on helix D of IL-13 or the equivalent in non-human IL-13useful in screening assays or for generating antibodies.

As indicated above, the GAG or polyanionic glycoconjugate target sitesin the AB loop and/or helix D can also be defined in terms of sulfatebinding motifs. Hence, the present invention contemplates sulfatebinding motifs in IL-13 selected from the list consisting of:

(i) amino acid residues Q22, Q24 and K25 in the AB loop of human IL-13;(ii) amino acid residues K97, D98, H102, K104, K105, R108, E109 and R111on helix D of human IL-13; and;(iii) amino acid residue Q22, Q24 and K25 in the AB loop and K97, D98,H102, K104, K105, R108, E109 and R111 of helix D of human IL-13.

As indicated above, these sites extend to derivatives and splicevariants of human IL-13 and the equivalent site in non-human IL-13homologs.

The present invention extends to IL-13 activity or function modifiers.Such molecules may be antagonists or agonists of IL-13 or IL-13interactions with a ligand such as a receptor or a receptor chain. Allsuch modifiers may be referred to herein as agents, therapeutics, drugs,molecules, active agents or similar term. Whilst agonists andantagonists are contemplated herein, antagonists are particularlyencompassed by the present invention.

Hence, the present invention is further directed to an isolated agentwhich modifies IL-13 activity or function the agent being capable ofinteracting with or antagonizing or agonizing binding of a GAG or apolyanionic glycoconjugate to a site on IL-13 comprising an amino acidin the AB loop and/or amino acids in helix D.

More particularly, the present invention provides an isolated agentwhich interacts with or antagonizes or agonizes binding of a GAG or apolyanionic glycoconjugate to a site on IL-13 comprising:

(i) a conformation of amino acid residues in the AB loop comprisingamino acid residues Q22, Q24 and K25;(ii) a conformation of amino acid residues on helix D comprising aminoacid residues K97, D98, H102, K104, K105, R108, E109 and R111; and;(iii) a conformation of amino acid residues comprising Q22, Q24 and K25in the AB loop and K97, D98, H102, K104, K105, R108, E109 and R111 onhelix D.

Yet another particular aspect of the present invention is directed to anisolated agent which interacts with or antagonizes or agonizes bindingof a GAG to a site on IL-13 comprising Q22, Q24 and K25 in the AB loopand K97, D98, H102, K104, K105, R108, E109 and R111 on helix D of humanIL-13 or the equivalent on a non-human IL-13 homolog.

The agents of the present invention may be identified, designed orselected by any number of means include in silico screening or modelingas well as x-ray crystallography, random screening, micro-arraytechnology and the like.

Hence, in one example, a computer-based method of in silico screening iscontemplated.

Screening or identifying a potential IL-13 modifier is suitablyfacilitated with the assistance of a computer programmed with software,which inter alia predicts binding of portions of a molecule to a GAG ora polyanionic glycoconjugate binding site on IL-13.

Thus, in another aspect, the present invention contemplates a computerprogram product for screening or designing an IL-13 modifier, theproduct comprising:

(a) code comprising coordinates or molecular shape defined by amino acidresidue numbers Q22, Q24 and K25 in the AB loop and K97, D98, H102,K104, K105, R108, E109 and R111 on helix D of human IL-13 or theequivalent in non-human IL-13 or a functional portion or region thereof;(b) code that screens known or theoretical ligands which might bind tothe coordinates or molecule shape; and(c) a computer readable medium that stores the codes.

In a related aspect, the invention extends to a computer for screeningor designing a IL-13 modifier, wherein the computer comprises:

(a) code comprising coordinates or molecular shape defined by amino acidresidue numbers Q22, Q24 and K25 in the AB loop and K97, D98, H102,K104, K105, R108, E109 and R111 on helix D of human IL-13 or theequivalent in non-human IL-13 or a functional portion or region thereof;(b) a working memory for storing instructions for processing themachine-readable data;(c) a central-processing unit coupled to said working memory and to saidmachine-readable data storage medium to provide known or theoreticalligands which might bind to the coordinates or molecule step; and(d) an output hardware coupled to said central processing unit forreceiving (a) and/or (b).

Another aspect provides a computer program product for screening ordesigning an IL-13 modifier, the product comprising:

(a) code comprising coordinates or molecular shape of:

-   -   (i) amino acid residues Q22, Q24 and K25 in the AB loop of human        IL-13 or its equivalent;    -   (ii) amino acid residue numbers K97, D98, H102, K104, K105,        R108, E109 and R111 or helix D of human IL-13 or their        equivalent; and    -   (iii) amino acid residue numbers Q22, Q24 and K25 in the AB loop        and K97, D98, H102, K104, K105, R108, E109 and R111 on helix D        of human IL-13 or their equivalents.        (b) code that screens known or theoretical ligands which might        bind to the coordinates or molecule shape; and        (c) a computer readable medium that stores the codes.

A further aspect is directed to a computer for screening or designing anIL-13 modifier, wherein the computer comprises:

(a) code comprising coordinates or molecular shape of:

-   -   (i) amino acid residues Q22, Q24 and K25 in the AB loop of IL-13        or its equivalent;    -   (ii) amino acid residues K97, D98, H102, K104, K105, R108, E109        and R111 on helix D of human IL-13 or its equivalent; and    -   (iii) amino acid residue Q22, Q24 and K25 in the AB loop and        K97, D98, H102, K104, K105, R108, E109 and R111 on helix D of        human IL-13 or their equivalents,        (b) a working memory for storing instructions for processing the        machine-readable data;        (c) a central-processing unit coupled to said working memory and        to the machine-readable data storage medium to provide known or        theoretical ligands which might bind to the coordinates or        molecule step; and        (d) an output hardware coupled to said central processing unit        for receiving (a) and/or (b).

In one version of these embodiments, a system including a computercomprising a central processing unit (“CPU”), a working memory which maybe, e.g. RAM (random-access memory) or “core” memory, mass storagememory (such as one or more disk drives or CD-ROM drives), one or morecathode-ray tube (“CRT”) display terminals, one or more keyboards, oneor more input lines, and one or more output lines, all of which areinterconnected by a conventional bidirectional system bus.

Input hardware, coupled to computer by input lines, may be implementedin a variety of ways. For example, machine-readable data of thisinvention may be inputted via the use of a modem or modems connected bya telephone line or dedicated data line. Alternatively or additionally,the input hardware may comprise CD. Alternatively, ROM drives or diskdrives in conjunction with display terminal, keyboard may also be usedas an input device.

Output hardware, coupled to computer by output lines, may similarly beimplemented by conventional devices. By way of example, output hardwaremay include CRT display terminal for displaying a syntheticpolynucleotide sequence or a synthetic polypeptide sequence as describedherein. Output hardware might also include a printer, so that hard copyoutput may be produced, or a disk drive, to store system output forlater use.

In operation, CPU coordinates the use of the various input and outputdevices coordinates data accesses from mass storage and accesses to andfrom working memory, and determines the sequence of data processingsteps. A number of programs may be used to process the machine readabledata of this invention.

The present invention further provides a magnetic data storage mediumwhich can be encoded with machine readable data, or set of instructions,for designing a synthetic molecule of the invention, which can becarried out by a system such as described above. Medium can be aconventional floppy diskette or hard disk, having a suitable substrate,which may be conventional, and a suitable coating, which may beconventional, on one or both sides, containing magnetic domains whosepolarity or orientation can be altered magnetically. Medium may alsohave an opening for receiving the spindle of a disk drive or other datastorage device. The magnetic domains of coating of medium are polarizedor oriented so as to encode in manner which may be conventional, machinereadable data such as that described herein.

The present invention also provides an optically readable data storagemedium which also can be encoded with such a machine-readable data, orset of instructions, for designing a synthetic molecule of theinvention, which can be carried out by a system. Medium can be aconventional compact disk read only memory (CD-ROM) or a rewritablemedium such as a magneto-optical disk, which is optically readable andmagneto-optically writable. Medium preferably has a suitable substrate,which may be conventional, and a suitable coating, which may beconventional, usually of one side of substrate.

A particular form of in silico screening is described in Raghuraman etal, J. Med. Chem. 49:3553-3562, 2006. In essence docking methods areemployed whereby various chemical groups are substituted in apentasaccharide in place of sulfate groups. This form of docking is alsoreferred to as combinatorial virtual screening.

The present invention provides, therefore, medicaments which modulatethe level of IL-13 activity or function. Reference to “medicaments”,“therapeutic molecules”, “agents”, “drugs”, “components” and“pharmaceuticals” may also be used to describe molecules which interactIL-13 and modify the activity.

The present invention further contemplates methods of screening fordrugs comprising, for example, contacting a candidate drug with a targetsite of IL-13 as identified herein. The screening procedure includesassay for the presence of a complex between the drug and a target siteas well as screening for any change in function. Cell-based screeningprocedures are also contemplated.

One form of assay involves competitive binding assays. In suchcompetitive binding assays, IL-13 is typically labeled. Free IL-13 isseparated from any putative complex and the amount of free (i.e.uncomplexed) label is a measure of the binding of the agent being testedto IL-13. One may also measure the amount of bound, rather than free,IL-13. It is also possible to label the putative agent rather than IL-13and to measure the amount of agent binding to IL-13 in the presence andin the absence of the drug being tested. Such compounds may inhibitIL-13 or may protect IL-13 from being inhibited or, alternatively, maypotentiate its inhibition.

A common cell based screening assay is to determine whether or not theagent being tested can inhibit cell proliferation that is induced byIL-13. TF-1 cells are an IL-13 responsive human cell line that iscommonly used for this purpose. Generally IL-13 is preincubated with theagent under test and then the mixture is added to TF-1 cells andproliferation is allowed to occur for 48 hours before the cell number isdetermined. If the agent binds to IL-13 so as to stop IL-13 frominteracting with its cell surface receptor chain complex, then theextent of cell proliferation in the presence of IL-13 mixed with theagent will be markedly lower than the extent of cell proliferationobtained when only the IL-13 is present. The same concentration of IL-13is used in both situations.

Cell based screening assays also include examining whether or not thedrug can modify the ability of leukocytes or leukocyte-like cell linesto traverse an endothelial cell layer grown in tissue culture. Theendothelial cell layer is grown on the upper surface of a porousmembrane supported on an insert added into the wells of a multi-welledplate. The leukocytes or leukocyte-like cell lines are added to theupper surface of the membrane in the presence of drug and the leukocytesare allowed to migrate across the endothelial cell layer and into thelower chamber. The extent of cell migration through the endothelial celllayer is monitored by means of a cell labeling dye, the level of dyeuptake being quantified using a plate reader.

In another method, GAG molecules are modified to give a drug which canmodulate the activity of IL-13. For example, sulfate groups on GAGs aremodified by being substituted with various non-ionic moieties. If thesulfate group on the GAG chain is replaced with the appropriate moietyindicated for that amino acid with which it interacts then this modifiedGAG structure may still bind, but bind with different affinity orantagonize non-modified GAG binding.

Such a modified GAG may be a suitable therapeutic candidate or a mimeticthereof may be designed or selected. Hence, the present inventioncontemplates GAG molecules, modified GAGs, GAG-like molecules,polyanionic glycoconjugates and composite GAGs as potential therapeutictargets or as template molecules to which suitable mimetics or homologsor structural equivalents may be designed. A GAG-like molecule includesa GAG-like composite structure having GAG or GAG-like components andnon-GAG components. The GAG or GAG-like molecules or GAG-like compositemolecules may be derived from naturally occurring heparin-like GAGs ormay be obtained by a process of chemical modification of a non-GAGpolysaccharide or may be a composite structure which may only in partcomprise a saccharide backbone.

These GAG or GAG-like oligosaccharides or GAG-like composite moleculesare generally regarded as semi-synthetic and are generated from eitherlarge polymeric polysaccharides as starting materials or discreteterta-, penta-, hexa-, septa- or octasaccharide starting materials. Thesemi-synthetic oligosaccharides may have varying degrees of chargedspecies, for example sulfates and/or phosphates. In addition, compoundsmay undergo a number of other modifications including modifications suchas the addition of side branches and phosphorylation of the GAGoligosaccharides. A library can also be made of the compositestructures. In this case there will be differences in the length of thenegatively charged units that resemble GAGs, differences in the lengthand composition of the linker that connects the negatively chargedregions that resemble GAGs and differences in the angles and flexibilityof the linker.

The linker structure of the GAG-like composite molecule is selectedfrom, but not limited to the following list comprising peptide,polypeptide or protein, chemical moiety, metal complexing agent,saturated or unsaturated fatty acid, lipid, dendrimer, saccharide,polyol, dextran, polyethylene glycol and branched or unbranched,saturated or unsaturated hydrocarbon chain.

The GAG-like oligosaccharides or the oligosaccharides of the GAG-likecomposite molecule comprise saccharides selected from, but not limitedto the following list consisting of glucuronic acid, iduronic acid,glucosamine, N-acetylglucosamine, galactosamine, mannose, mannan,dextran, glucose, galactose, fructose, sucrose, fucose, heptulose,pentose, xylose, arabinose, manuronic acid, anhydrogalactose andguluronic acid. The oligosaccharide may be obtained by a process ofchemical modification comprising enzymatic or chemical digestion of apolysaccharide of populations of polysaccharides and a step selectedfrom the list comprising deacetylation; sulfation; desulfation;phosphorylationa and attachment of side chains. The oligosaccharides maycomprise a sulphated and/or phosphorylated form thereof and/oracetylated form thereof or contain alkyl ether derivatives comprisingmethyl, ethyl, propyl or butyl. The GAG molecules include heparin,heparan sulfate and pentosan polysulfate (PPS). Hence, heparin, heparansulfate, PPS and fractions or derivatives thereof are contemplatedherein to bind to IL-13 or to inhibit IL-13/IL-13R interaction.

Truncation of the polysaccharides into oligosaccharides can be achievedvia a number of mechanisms. The methods employed to truncate thepolysaccharide include enzymatic, chemical, thermal and ultrasonicprotocols such as described by Alban and Franz, Biomacromolecules 2:354, 2001.

The termini of the GAG-like oligosaccharides or the oligosaccharides ofthe GAG-like composite molecule comprises4-deoxy-L-threo-hex-4-enopyranosyluronic acid. The GAG-likeoligosaccharide or the oligosaccharides of the GAG-like compositemolecule contains a terminal glucosamine which is modified as a resultof nitrous acid treatment and is selected from the group consisting of2,5-anhydro-D-mannitol and 2,5-anhydro-D-mannose.

The saccharide unit of the GAG-like oligosaccharides or theoligosaccharides of the GAG-like composite molecule has one or moremono-, di-, or tri-saccharide units appended to hydroxyl groups in the 6position to give a branched structure.

The present invention is further directed to compositions such aspharmaceutical compositions comprising the IL-13 modifiers hereincontemplated.

The terms “modifier”, “compound”, “active agent”, “pharmacologicallyactive agent”, “medicament”, “active” and “drug” are usedinterchangeably herein to refer to a molecule that induces a desiredpharmacological and/or physiological effect and in particularantagonizes or agonizes IL-13 activity or function. The terms alsoencompass pharmaceutically acceptable and pharmacologically activeingredients of those active agents contemplated herein including but notlimited to salts, esters, amides, prodrugs, active metabolites, analogsand the like. When the terms “modifier”, “compound”, “active agent”,“pharmacologically active agent”, “medicament”, “active” and “drug” areused, then it is to be understood that this includes the active agentper se as well as pharmaceutically acceptable, pharmacologically activesalts, esters, amides, prodrugs, metabolites, analogs, etc. The term“compound” is not to be construed as a chemical compound only butextends to peptides, polypeptides and proteins and chemical analogsthereof. The modifiers identified or screened in accordance with thepresent invention are proposed to be useful in modulating inflammatoryprocesses including inhibiting inflammation, inhibiting fibrosis,inhibiting growth of cancer cells and promoting or inhibiting stem cellproliferation and/or differentiation. The modifier includes a GAGmolecule, heparin and heparan sulfate as well as fractions orderivatives thereof, or GAG-like molecules like PPS or other anionicpolysaccharides.

The compounds, therefore, have an effect on reducing or preventing ortreating inflammatory conditions. Reference to a “compound”, “activeagent”, “pharmacologically active agent”, “medicament”, “active” and“drug” includes combinations of two or more actives such as one or moreinhibitors and/or potentiators of IL-13 function or activity. A“combination” also includes a two-part or more such as a multi-partpharmaceutical composition where the agents are provided separately andgiven or dispensed separately or admixed together prior to dispensation.

The terms “effective amount” and “therapeutically effective amount” ofan agent as used herein mean a sufficient amount of the agent to providethe desired therapeutic or physiological effect. Undesirable effects,e.g. side effects, are sometimes manifested along with the desiredtherapeutic effect; hence, a practitioner balances the potentialbenefits against the potential risks in determining what is anappropriate “effective amount”. The exact amount required will vary fromsubject to subject, depending on the species, age and general conditionof the subject, mode of administration and the like. Thus, it may not bepossible to specify an exact “effective amount”. However, an appropriate“effective amount” in any individual case may be determined by one ofordinary skill in the art using only routine experimentation.

By “pharmaceutically acceptable” carrier, excipient or diluent is meanta pharmaceutical vehicle comprised of a material that is notbiologically or otherwise undesirable, i.e. the material may beadministered to a subject along with the selected active agent withoutcausing any or a substantial adverse reaction. Carriers may includeexcipients and other additives such as diluents, detergents, coloringagents, wetting or emulsifying agents, pH buffering agents,preservatives, and the like.

Similarly, a “pharmacologically acceptable” salt, ester, emide, prodrugor derivative of a compound as provided herein is a salt, ester, amide,prodrug or derivative that this not biologically or otherwiseundesirable.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble), sterile powders for theextemporaneous preparation of sterile injectable solutions and inhalableforms. Such forms are preferably stable under the conditions ofmanufacture and storage and are generally preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dilution medium comprising, for example,water, ethanol, polyol (for example, glycerol, propylene glycol andliquid polyethylene glycol, and the like), suitable mixtures thereof andvegetable oils. The proper fluidity can be maintained, for example, bythe use of superfactants. The preventions of the action ofmicroorganisms can be brought about by various anti-bacterial andanti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thirmerosal and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activeingredients in the required amount in the appropriate solvent with theactive ingredient and optionally other active ingredients as required,followed by sterilization or at least a process to reduce contaminatingviruses, bacteria or other biological entities to acceptable levels foradministration to a human or animal subject. In the case of sterilepowders for the preparation of sterile injectable solutions, suitablemethods of preparation include vacuum drying and the freeze-dryingtechnique that yields a powder of active ingredient plus anyadditionally desired ingredient.

When the active ingredient is suitably protected, it may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets. For oral therapeuticadministration, the active ingredient may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers and the like.Such compositions and preparations should contain at least 1% by weightof active compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 5 toabout 80% of the weight of the unit. The amount of active compound insuch therapeutically useful compositions is such that a suitable dosagewill be obtained. Preferred compositions or preparations according tothe present invention are prepared so that an oral dosage unit formcontains between about 0.1 μg and 200 mg of active compound. Alternativedosage amounts include from about 1 μg to about 1000 mg and from about10 μg to about 500 mg. These dosages may be per individual or per kgbody weight. Administration may be per second, minute, hour, day, week,month or year.

The tablets, troches, pills and capsules and the like may also containthe components as listed hereafter. A binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added. When thedosage unit form is a capsule, it may contain, in addition to materialsof the above type, a liquid carrier. Various other materials may bepresent as coatings or to otherwise modify the physical form of thedosage unit. For instance, tablets, pills or capsules may be coated withshellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavour. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound(s) may be incorporated intosustained-release preparations and formulations.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, anti-bacterial and anti-fungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art and except insofar as any conventional media or agent isincompatible with the active ingredient, their use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The composition may also be formulated for local or topicaladministration. Techniques formulation and administration may be foundin “Remington's Pharmaceutical Sciences”, Mack Publishing Co., EastonPa., 16th edition, 1980, Ed. By Arthur Osol. Thus, for local or topicaladministration, the subject compositions may be formulated in anysuitable manner, including, but not limited to, creams, gels, oils,ointments, solutions, suspensions, powders, mists or aerosols. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art and include, but are not restricted to, benzalkoniumchloride, digitonin, dihydrocytochalasin B, and capric acid.

The compositions of the subject invention in the form of lotions, creamsor gels may contain acceptable diluents or carriers to impart thedesired texture, consistency, viscosity and appearance. Acceptablediluents and carriers are familiar to those skilled in the art andinclude, but are not restricted to, ethoxylated and nonethoxylatedsurfactants, fatty alcohols, fatty acids, hydrocarbon oils (such as palmoil, coconut oil, and mineral oil), cocoa butter waxes, silicon oils,buffering agents, cellulose derivatives, emulsifying agents such asnon-ionic organic and inorganic bases, preserving agents, wax esters,steroid alcohols, triglyceride esters, phospholipids such as lecithinand cephalin, polyhydric alcohol esters, fatty alcohol esters,hydrophilic lanolin derivatives, and hydrophilic beeswax derivatives.

In one particularly preferred embodiment, the present inventioncontemplates an inhalant pharmaceutical composition.

The terms “treating” and “treatment” as used herein refer to reductionin severity and/or frequency of symptoms, elimination of symptoms and/orunderlying cause, prevention of the occurrence of symptoms and/or theirunderlying cause, and improvement or remediation of damage. Thus, forexample, “treating” a patient involves prevention of a particulardisorder or adverse physiological event in a susceptible individual aswell as treatment of a clinically symptomatic individual by inhibitingor causing regression of an condition or disorder such as aninflammatory condition or disorder. Generally, such a condition ordisorder is an inflammatory response or mediates or facilitates aninflammatory response or is a downstream product of an inflammatoryresponse. Thus, for example, the present method of “treating” a patientwith an inflammatory condition or with a propensity for one to developencompasses both prevention of the condition, disease or disorder aswell as treating the condition, disease or disorder.

“Patient” as used herein refers to an animal, particularly a mammal andmore particularly human who can benefit from the pharmaceuticalformulations and methods of the present invention. There is nolimitation on the type of animal that could benefit from the presentlydescribed pharmaceutical formulations and methods. A patient regardlessof whether a human or non-human animal may be referred to as anindividual, subject, animal, host or recipient. The compounds andmethods of the present invention have applications in human medicine,veterinary medicine as well as in general, domestic or wild animalhusbandry. For convenience, an “animal” includes an avian species suchas a poultry bird, an aviary bird or game bird.

Particular animals are humans or other primates, livestock animals,laboratory test animals, companion animals or captive wild animals. Ahuman is the most preferred target.

Examples of laboratory test animals include mice, rats, rabbits, guineapigs and hamsters. Rabbits and rodent animals, such as rats and mice,provide a convenient test system or animal model. Livestock animalsinclude sheep, cows, pigs, goats, horses and donkeys. Non-mammaliananimals such as avian species, zebrafish, amphibians (including canetoads) and Drosophila species such as Drosophila melanogaster are alsocontemplated. Instead of a live animal model, a test system may alsocomprise a tissue culture system.

The present invention also contemplates the use of a GAG or polyanionicglycoconjugate binding site on IL-13 in the manufacture of a medicamentfor modulating a physiological process such as inflammatory processes ina subject.

As indicated above, the GAG or polyanioinc glycoconjugate binding siteis selected from the list consisting of an amino acid in the AB loop, anamino acid on helix D and amino acids in the AB loop and on helix D.

More particularly, the GAG binding site is selected from the listconsisting of:

(i) a conformation of amino acid residues comprising amino acid residuesQ22, Q24 and K25 in the AB loop on human IL-13 or its equivalent;(ii) a conformation of amino acid residues comprising K97, D98, H102,K104, K105, R108, E109 and R111 on helix D of human IL-13 or theirequivalents; and(iii) a conformation of amino acid residues comprising Q22, Q24 and K25in the AB loop and K97, D98, H102, K104, K105, R108, E109 and R111 onhelix D of human IL-13 or their equivalents.

The IL-13 activity or function modifiers are particularly useful in thetreatment of a range of inflammatory conditions such as allergicrhinitis, chronic obstructive pulmonary disease (COPD), emphysema,asthma, Crohn's disease, ulcerative colitis, inflammatory bowel disease,systemic lupus erythematosus, rheumatoid arthritis, Alzheimer's disease,type I diabetes, gingivitis, eczema (atoptic dermatitis), psoriaticarthritis, tendinitis and multiple sclerosis. Asthma and allergicrhinitis are particular conditions encompassed herein. The IL-13activity or function modifiers are also useful in the treatment of otherdiseases to which IL-13 contributes including fibrosis and variouscancers, for example B chronic lymphocytic leukemia (B-CLL), Hodgkin'sdisease, where tumor growth/protection from apoptosis is promoted byIL-13, and other cancers in which IL-13 appears to antagonize tumorimmunosurveillance (Wynn, 2003 supra). The link between IL-13 andfibrosis suggests that IL-13 antagonists may also be effective in avariety of situations where chronic exposure to IL-13 triggers excessivehealing, tissue remodeling, or the formation of destructive tissuepathology in situations like idiopathic pulmonary fibrosis, chronicgraft rejection, bleomycin-induced pulmonary fibrosis, progressivesystemic sclerosis, radiation-induced pulmonary fibrosis and hepaticfibrosis.

Hence, the present invention also contemplates a method of treatment orprophylaxis of a physiological process including an inflammatory processin a subject the method comprising administering to a subject an IL-13activity or function modifier which binds or interacts with a GAGbinding site on IL-13 selected from the list consisting of the AB loopand helix D.

In a particular embodiment, the GAG or polyanionic glycoconjugatebinding site is selected from the list consisting of:

(i) a conformation of amino acid residues comprising Q22, Q24 and K25 inthe AB loop of human IL-13 or its equivalent;(ii) a conformation of amino acid residues comprising K97, D98, H102,K104, K105, R108, E109 and R111 in helix D of human IL-13 or theirequivalents;(iii) a conformation of amino acid residues comprising Q22, Q24 and K25in the AB loop and K97, D98, H102, K104, K105, R108, E109 and R111 onhelix D of human IL-13 or their equivalents.

The present invention is further described by the following non-limitingExamples. In these Examples, methods described below are employed.

Homology Modeling of IL-13

Sequences of the human, mouse, rat and porcine IL-13, as well as variousalternatively spliced isoforms, are retrieved from the SWISS-PROTprotein sequence database (Boeckmann et al, Nucleic Acids Research31:365-370, 2003). Multiple sequence alignment are performed withCLUSTALW (Thompson et al, Nucleic Acids Research 22:4673-4680, 1994)using BLOSUM (BLOcks of Amino Acid Substitution Matrix) matrices inorder to quantify the sequence similarity between individual subunits ofIL-13 in different species and in the different isoforms. Initially, aPSI-BLAST (Altschul et al, Nucleic Acids Research 25:3389-3402, 1997)search against the Protein Data Bank is performed in order to findsequences that were homologous with human IL-13, so that proteins ofknown structure are identified and used as a global template forstandard homology modeling. Additionally, different sequence andsecondary structure prediction algorithms (PredictProtein [Rost et al,Nucleic Acid Research 32:W321-326, 2004] and PSIPRED [Bryson et al,Nucleic Acid Research 33:W36-38, 2005]) are used to predict the residuescomprising the different secondary structures. The AB loop and helix Dsequences, as classified by SWISSPROT are submitted to the foldrecognition services Phyre (Kelley et al, J Mol Biol 299(2):499-520,2000) [a successor algorithm of 3D-PSSM] and CBS Meta Server (Douguetand Labesse, Bioinformatics 17:752-753, 2001). Alignments are performedof the AB loop and helix D region of IL-13 with crystal structures ofknown AB loop and helix D regions using LALIGN/PLALIGN (Pearson andLipman, PNAS 85(8):2444-2448, 1988). This allows the inclusion of newstructural information available from time to time in the subsequentstages of the modeling studies. The statistical significance of analignment is computed by aligning the two sequences and then shufflingthe second sequence between 200 and 1000 times using the PRSS module(Pearson 1988 supra).

Structure construction, assignment of disulfide bridges, optimizationand visualization are carried out using the molecular modeling packageDS Modeling 1.7 (Accelrys, Inc.) Loops are built using the loop modelingprotocol implemented in MODELLER (Fiser et al, Protein Sci9(9):1753-1773, 2000; Sali and Blundell, J Mol Biol 234(3):779-815,1993). Essential hydrogen and charges are added to the structure. Thestructural quality of the resultant protein structure is tested usingPROCHECK (Laskowski et al, Journal of Applied Crystallography26(2):283-291, 1993), Eva123D (Douguet and Labesse 2001 supra) andVerify3D (Douguet and Labesse 2001 supra). Electrostatis potentialcalculations are done using the DELPHI program (Gilson and Honig, Nature330(6143):84-86, 1987) implemented in DS Modeling 1.7 (Accelrys, Inc.)using the atomic partial charges assigned by CHARM with a proteininterior dielectric constant of 4, a solvent dielectric constant of 80and an ionic strength of 0.145M.

Alternatively high-resolution solution structures of human IL-13 havebeen determined by multidimensional NMR and have been deposited in theRCSB Protein Data Bank, PDB ID: 1ijz or 1iko (Moy et al, J. Mol. Biol.310:219-230, 2001) or PDB ID: 1GA3 (Eisenmesser, et al, J. Mol. Biol.310:231-241, 2001). These structures can be used instead of the homologymodeling approach. The PDB is surveyed for sulfate binding motifs usingBLAST searches for short overlapping segments for AB loops and helix Ddomains.

PatchDock (Schneidman-Duhovny et al, Nucleic Acids Res 33:W363-367,2005; Schneidman-Duhovny et al, Proteins 52(1):107-112, 2003) is used todock heparin and other GAG fragments to the entire IL-13 model.PatchDock is a fast geometry-based molecular docking algorithm thatworks by optimizing shape complementarity (hence, it is not an energygrid-based method). No constraints are used to define the binding sitein order to allow the program to explore the entire surface of IL-13 andfind appropriate interaction regions using an RMSD clustering in orderto reduce the number of potential binding modes (Schneidman-Duhovny etal 2005 supra).

Most three-dimensional X-ray structures of GAG-protein complexesdetermined so far involve relative small oligosaccharides (di- tohexasaccharides) of varying affinity for their protein targets. In orderto determine the minimal length of the heparin fragments required forbinding to the AB loop/helix D domains of IL-13, docking simulations areperformed with di- and pentasaccharides. The structure of the heparinpentasaccharide can be obtained from the crystal structure of annexin A2complexed with an unsaturated hexasaccharide in PDB code 2HYV. Since noelectron density is observed for the sixth saccharide residue (Shao etal, J Biol Chem 281(42):31689-31695, 2006), the pentassacharide isextracted directly from the structure. The residue at the non-reducingend of the heparin pentasaccharide is modified from UA2S by the additionof a hydrogen to the double bond between C-4 and C-5 to create a 4-deoxyIdoA2S residue (4dIdoA2S). This modeled pentasaccharide consists of4dIdoA2S (1→4)GlcNS6 S(1→4)IdoA2S (1→4)GlcNS6S (1→4)IdoA2S. The pyranoserings of the glucosamine residues adopt a ⁴C₁ chair conformation whereasiduronic acids can adopt either a ¹C₄ chair or a ²S_(o) skew-boatconformation.

The structure of the disaccharide (IdoA2S (1→4)GlcNS6S)can be extractedfrom the reported NMR structures of a heparin dodecassacharide fragment(PDB structure 1HPN) [Mulloy et al, Biochem J293(3):849-858, 1993].

Docking of the dermatan sulfate (DS) tetrasaccharide (PDB code 1HM2) anda pentasaccharide extracted from chondroitin-4-sulfate (CS, PDB code1C4S) is also performed. There is no crystal structure available for thedermatan sulfate pentasaccharide. The modeled DS tetrasaccharideconsisted of IdoA(1→3)GalNAc4S(1→4)IdoA(1→3)GalNAc4S and the modeled CSconsisted of GlcA(1→3)GalNAc4S(1→4)GlcA(1→3)GalNAc4S(1→4)GlcA Hydrogenatoms are added to these oligosccharides and the resultant structuresare energy minimized in order to optimize the orientation of rotatablegroups. The surface area, atomic contact energy and the binding scorecomputed by PatchDock for the heparin pentasaccharide are extracted.

Further docking simulations are carried out using the program AutoDock3.0 (Morris et al, Journal of Computational Chemistry 19(14):1639-1662,1998). This program allows for flexibility in the ligand structure butuses a rigid body approximation for the protein receptor in order tospeed up the calculation. AutoDock Tools (ADT) (Sanner and Python, J MolGraph Model 17(1):57-61, 1999) are used to prepare the IL-13 molecule byadding appropriate hydrogens, partial atomic charges and salvationparameters. Ligand rotatable bonds for all docked ligands are definedusing the AutoTors module of AutoDock. The ligands were atom-typemanually to ensure that they complied with the carbohydrate forcefieldin AMBER (Weiner et al, J. Am. Chem. Soc. 106(3):765-784, 1984). Theligands are energy minimized in order to optimize the orientation of itshydrogen atoms. A grid spacing of 0.37 Å and a distance-dependentdielectric constant of 4.0 (as defined by Mehler and Somajer, ProteinEng 4(8):903-910, 1991) are used for the binding energy calculations,covering the putative binding site surface. Using AutoDock's Lamarckiangenetic algorithm, heparin fragments are subjected to search runs usinga population of individuals. The grid box is defined with a constantgrid spacing of 0.37 Å around each heparin fragment using the bindingposes obtained from PatchDock with respect to the AB loop and helix Ddomains of IL-13.

Due to the flexibility and size of the di- and pentasaccharides ofheparin, the number of energy evaluations and the size of the geneticpopulation are optimized to ensure convergence of the calculatedenergies, starting with a minimum of 5×10⁶ and a maximum of 50×10⁶energy evaluations, as reported for blind docking (Hetenyi and van derSpoel, Protein Sci 11(7):1729-1737, 2002). Cluster analysis is performedon the resulting binding poses using a root mean square deviation (RMSD)tolerance of 1.0 Å. Since AUTODOCK cannot handle more than 32 rotatablebonds, the docking of the heparin fragments is performed keeping thehydroxyl groups fixed. The lowest docking energy binding scores of thedisaccharides with full rotational freedom of their hydroxyl groups areverified to be similar to those obtained with the hydroxyl groups keptfixed, confirming that the initial orientation of the hydroxyl groups isappropriate for interactions with IL-13.

Combinatorial virtual screening is conducted by any number of meansincluding the method of Raghuraman et al, 2006 supra.

Hence, a combinatorial virtual screening approach is proposed herein forpredicting high specificity heparin/heparan sulphate sequences using theAB loop and helix D domains of IL-13-heparin pentasaccharide. Heparansulphate/heparin pentasaccharide is simulated keeping theinter-glycosidic bond angles constant at the mean of the known solutionvalues, irrespective of their sequence. Molecular docking using geneticalgorithm (GA) implemented in softwares such as AutoDock and GOLD withrestrained inter-glycosidic torsions and intra-ring conformations, butflexible substituents at the 2-, 4-, and 6-positions. Cluster analysisis performed on the resulting binding poses using an RMSD tolerance of2.5 Å from the reference pose (docked pentasaccharide 2HYV).

The pentasaccharide sequence 2HYV, or sequence ABCDE[IdoA2S(1→4)GlcNS6S(1→4)IdoA2S(1→4)GlcNS6S(1→4)IdoA2S], is docked to ABloop and helix D domains of IL-13 using AutoDock. The glucosamineresidues are modelled in a ⁴C₁ chair conformation whereas the iduronicacid can exist either in the ²S_(O) or C₄ conformations. The ligands areatom-typed manually to ensure that they complied with the carbohydrateforcefield in AMBER. In SYBYL, the atom type of sulfur and oxygen atomsin SO₃ groups is modified to ²S_(o) and ²O_(co), respectively, and thebond type between these atoms was modified to aromatic bond. Hydrogenatoms, absent in the PDB structure, are added in SYBYL, and theresultant structure is minimized to optimize geometry of hydrogen atomsonly (no change in non-H atoms). A grid spacing of 0.37 Å and adistance-dependent dielectric constant of 4.0 (as defined by Mehler andSolmajer, 1991 supra) are used for the binding energy calculations,covering the putative binding site surface. Using AutoDock's Lamarckiangenetic algorithm, heparin fragments are subjected to search runs usinga population of individuals with a maximum of 50×10⁶ energy evaluations.

Docking is driven by the AutoDock scoring function. This free energyestimate of binding and docking energy including clustering is used torank the final docked solutions.

The natural pentasaccharide structure docked to the AB loop/Helix D ofIL-13 can be used as a template for the generation of GAG mimetics. GAGmimetics can consist of structures in which the GAG backbone is retainedbut the sulfates are variously replaced with particular structuralgroups, these structural groups include, but are not limited to,hypoxanthine, phenyl, amide and uracil groups. For each of these GAGmimetics consisting of structural modifications to the naturalpentasaccharide sequence, minimization is performed with constraintsthat retain the sugar conformations. Because IdoA residues in heparincan exist either in the ²S_(O) or ¹C₄ conformations, each IdoA residueshould be modeled explicitly in these two different states. The dockingof these sequences can be carried out according to the protocoldescribed above for the natural pentasaccharide.

A combinatorial library of heparin pentasaccharides is generated fordocking onto AB loop/helix D of IL-13. The library considers theconformational flexibility of IdoA residue domains through inclusion ofboth ¹C₄ and ²S_(O) conformations. Thus, the combinatorial libraryconsists of possible combinations of unique heparin pentasaccharidesbuilt with the average backbone geometry (similar to the naturalpentasaccharide) in an automated manner. In addition, GAG mimetics maybe generated by using a different structural backbone to display thesulphate residues in a fashion so as to facilitate binding to theGAG-binding site on IL-13 (GAG-like oligosaccharides).

The GAG mimetic may also comprise a GAG-like-composite structure and beclassified as a polyanionic glycoconjugate. In a particular aspect, thesaccharide structure comprises two or more high charged (e.g. sulfatedor phosphorylated) disaccharides or trisaccharides or tetrasaccharidesor pentasaccharides or hexasaccharides or heptasaccharides oroctasaccharides or any combination of these saccharides separated by alinker or linkers. The linker is not necessarily based on a GAG-likebackbone. Rather, a linker such as an alkyl chain or a polyol structure,or polyethylene glycol is preferable. Further, it is not necessary forthe highly charged saccharides to be based on GAG structures. TheGAG-like oligosaccharides or the oligosaccharides of the GAG-likecomposite molecule may comprise saccharides selected from, but notlimited to the following list consisting of glucuronic acid, iduronicacid, glucosamine, N-acetylglucosamine, galactosamine, mannose, mannan,dextran, glucose, galactose, fructose, sucrose, fucose, heptulose,pentose, xylose, arabinose, manuronic acid, anhydrogalactose andguluronic acid. The oligosaccharide may be obtained by a process ofchemical modification comprising enzymatic or chemical digestion of apolysaccharide of populations of polysaccharides and a step selectedfrom the list comprising deacetylation; sulfation; desulfation;phosphorylationa and attachment of side chains. The oligosaccharides maycomprise a sulphated and/or phosphorylation form thereof and/oracetylated form thereof or contain alkyl ether derivatives comprisingmethyl, ethyl, propyl or butyl.

Truncation of the polysaccharides into oligosaccharides can be achievedvia a number of mechanisms. The methods employed to truncate thepolysaccharide include enzymatic, chemical, thermal and ultrasonicprotocols such as described by Alban and Franz, Biomacromolecules 2:354, 2001.

The termini of the GAG-like oligosaccharides or the oligosaccharides ofthe GAG-like composite molecule comprises4-deoxy-L-threo-hex-4-enopyranosyluronic acid. The GAG-likeoligosaccharide or the oligosaccharides of the GAG-like compositemolecule contains a terminal glucosamine which is modified as a resultof nitrous acid treatment and is selected from the group consisting of2,5-anhydro-D-mannitol and 2,5-anhydro-D-mannose.

In an alternate embodiment of the present invention, GAGoligosaccharides are generated by the method comprising sizefractionating a population of heparin or heparan sulfate molecules orother polymers such as K5 polysaccharide, chitin or chitosan to generatenon-full length fraction which interact with a ligand. Heparin comprisesmixtures of glucuronic and iduronic acids in some of the disaccharideunits and varying degrees of sulfation. Fractionation may be by anyconvenient means such as by gel filtration column and is based ondifferent length saccharide chains generally but not exclusively from adegree of polymerization (DP)4 to about a DP20 such as DP5, DP6, DP7,DP8, DP9, DP10, DP11, DP12, DP13, DP14, DP15, DP16, DP17, DP18 or DP19.Another form of separation is on the basis of extent of sulfation. Theseparation may also be based on a combination of these two parameters.

Another aspect of the present invention is the provision of novelphosphorylated GAG oligosaccharides. These molecules are generated bythe inclusion of a phosphorylation step when generating the library ofGAG oligosaccharides. This phosphorylation step and any associateddesulfation steps that may be necessary, is performed on both thesemi-synthetic oligosaccharides derived from polymers such as E. coli K5polymer, chitin or chitosan, and oligosaccharides produced byfractionation of GAGs such as heparin or heparan sulfate.

The 6-O sulfate is the easiest O-sulfate to hydrolyse, giving a way toaccess free 6-OH. The free 6-OH is then phosphorylated to give the 6-Ophosphate. The sulfate and phosphate esters have been shown to beequipotent in many compounds although in others phosphorylation changesthe activity. It is also possible to N-phosphorylate the glucosamineresidue.

The selective phosphorylation of a hydroxyl group is readily achievedusing the phosphoramidate-oxidation method (Vieira de Almeida et al,Tetrahedron 55: 7251-7270, 1999; Dubreuil et al, Tetrahedron 55:7573-7582, 1999 and references cited therein) This method has beenwidely employed for the formation of inositol phosphates, nucleotidesand oligonucleotides. Alternatively, several other more rapid methodsfor the introduction of a phosphate group could be employed such asphosphoryl oxychloride in the presence of pyridine followed by aqueoushydrolysis. It may also be possible to enzymatically phosphorylate theseoligosaccharides through the agency of a promiscuous hexose kinaseenzyme.

The saccharide unit of the GAG-like oligosaccharides or theoligosaccharides of the GAG-like composite molecule has one or moremono-, di-, or tri-saccharide units appended to hydroxyl groups in the 6position to give a branched structure.

Each oligosaccharide pool, as produced according to the methodsdescribed above, is then tested for its ability to interact or bind toIL-13 or IL-13R. This may be performed experimentally or in silico.

The heparan sulfate/heparin pentasaccharide combinatorial library ismade for screening all possible sequences using GA iterationsimplemented in AutoDock as described above. Free energy and dockingenergy scoring functions (see Table 2) in AutoDock aids in identifyingthe most promising sequences that have a relatively high bindingaffinity. The second step can consist of clustering these sequences withrespect to the top two ranked solutions within 2.5 Å RMSD as compared tothe natural pentasaccharide.

TABLE 2 Energy scouring functions Estimated Free Energy of Binding (1) +(3) Docked Energy (1) + (2) (1) Final Intermolecular Energy in kcal/mol(2) Final Internal Energy of Ligand in kcal/mol (3) Torsional Freeenergy in kcal/mol

The approach facilitates the extraction of a pharmacophore, the keyinteractions to identify individual GAG sequences or GAG mimetics withhigh specificity. Energy scoring functions combined with a map of RMSDof atoms compared to the natural pentasaccharide can be readily createdfrom the combinatorial library screening experiments to identify GAGsequences or GAG mimetic sequences which can define the pharmacophore.Thus, this approach is also useful for designing therapeutically usefulmolecules.

The interaction with a ligand may be measured experimentally by anyconvenient means such as gel retardation, filter retardation, affinityco-electrophoresis, bioluminescent resonance energy transfer (BRET)assays, fluoresence resonance energy transfer (FRET) assays,fluorescence polarization (FP) assays, scintillation proximity assays orimmobilization to biochips or other surfaces including those coupledwith mass spectrometric detection.

The latter may be accomplished by first immobilizing the GAGoligosaccharide or heparin or GAG-like polyanionic polysaccharideapolyanionic glycoconjugate to a chip and then adding the IL-13 in thefluid phase. Alternatively, IL-13 may be immobilized to a chip and usedto screen for the ability of a GAG oligosaccharide, or heparin, orGAG-like polyanionic polysaccharide or a polyanionic glycoconjugate tobind thereto.

Yet another alternative is to immobilize a GAG, such as heparin, to asolid support and then screen for the ability of a polyanionicglycoconjugate, GAG-likepolyanionic polysaccharide or a GAGoligosaccharide, produced according to the methods above, to inhibitbinding of IL-13 to the immobilized heparin.

Accordingly, a particularly useful assay is to admix IL-13 and thepolyanionic glycoconjugate or a GAG oligosaccharide or GAG-likepolyanionic polysaccharide and screen for the ability for of thepolyanionic glycoconjugate, GAG oligosaccharide or GAG-like polyanionicpolysaccharide to inhibit binding of IL-13 to a GAG (e.g. heparin orheparan sulfate) bound to a chip.

A common cell based assay involves testing whether or not thepolyanionic glycoconjugate or a GAG oligosaccharide or a GAG-likepolyanionic polysaccharide can inhibit cell proliferation that isinduced by IL-13. TF-1 cells are an IL-13 responsive human cell linethat is commonly used. IL-13 is preincubated with the agent under testand then the mixture is added to TF-1 cells and proliferation is allowedto occur for 48 hours before the cell number is determined. If the agentbinds to IL-13 so as to stop IL-13 from interacting with its cellsurface receptor chain complex, then the extent of cell proliferation inthe presence of IL-13 mixed with the agent will be markedly lower thanthe extent of cell proliferation obtained when only the IL-13 ispresent. The same concentration of IL-13 is used in both situations.

Example 1 Sequence and Structure in Heparin and Heparan Sulfate

The most intensively studied and best understood sequence ofmonosaccharide residues in heparin is an unusual pentasaccharide whichis the minimum requirement for high affinity to antithrombin. It is thissequence which accounts for the high anticoagulant potency of heparin,and hence its use as an antithrombotic agent; the essentialpentasaccharide has been prepared synthetically and is itself used as adrug (Petitou and van Boeckel, Angew. Chem. Int. Edit. 43:3118, 2004).When it became clear that heparin, as a model compound for heparansulfate, was capable of physiologically important interactions withother classes of protein, such as the fibroblast growth factors(Mohammadi et al, Curr. Opin. Struct. Biol. 15:506, 2005), the exampleof the antithrombin-binding sequence led to a search for other, equallyspecific sequences in either heparin or heparan sulfate which wouldconfer particular affinity for any given binding partner. This searchfor specificity of a high order has on the whole been unsuccessful, anda recent study of structures which are capable of potentiatingfibroblast growth factor (FGF)-mediated cell growth has concluded thatheparan sulfate fine structure may be less influential than haspreviously been supposed (Kreuger and Spillmann, J. Cell Biol 174:323,2006).

Heparan sulfate is often represented, and imagined, in terms ofsequences, rather than three-dimensional structures (Mulloy, Anais Acad.Bras. Cienc. 77:651, 2005). The crystal structure of FGF-1 (2axm.pdb)complexed with a heparin oligosaccharide (Stauber et al, Proc. Natl.Acad. Sci. USA 97:49, 2000) shows clearly that the pattern of sulfategroups interacting with the protein can be formed in two ways, involvingclusters of sulfate and carboxylate substituents on either side of thepolysaccharide chain. Two separate molecules of FGF-1, aligned inopposite directions along the heparin chain, each interact with acluster of three sulfates, part of a second cluster, and the carboxylatebetween the two clusters. The charge-based interactions, between theacidic substituents on the polysaccharide and basic residues on thesurface of the protein, usually dominate the interface, and the detailednature of the sugar backbone carrying the substituents is much lessimportant, so long as it presents the substituents in an appropriatethree-dimensional pattern. This “pseudo-symmetry”, in which theunderlying asymmetry of the sugar backbone is hidden by the almostsymmetrical arrangement of bulky and highly charged substituents, is acomplicating factor in the interpretation of heparan sulfate sequencerequirements for affinity with different proteins. Another such factoris the finding that, for most interactions between heparin and proteins,substitution with additional sulfate groups does not decrease affinity.Bearing in mind both factors together, a simplistic calculationindicates 31 different pentasaccharide sequences (starting and endingwith glucosamine) which will contain a single FGF-1 binding motifLeaving the motif on one side of the molecule undisturbed, and assumingthat any or all of the four remaining sulfated positions may or may notbe occupied, 16 (2⁴) different possible compounds can be defined.Repeating this exercise for the second side gives 31 possible sequencesin all (not counting the fully sulfated compound twice). Such a sequenceis more likely to occur in highly sulphated regions of thepolysaccharide. Potentiation of growth factor activity is more complexthan simple affinity for the growth factor itself, and it is clear thatthe requirements for functional interaction with the growthfactor/receptor complex are not the same as for the growth factor alone(Ostrovsky et al, J. Biol. Chem. 277:2444, 2002); still, the search fora 3-D pattern is more likely to be successful than for a specificsequence.

Example 2 Drug Discovery Techniques

Molecular modeling techniques, in particular those in which a smallmolecule is docked into its binding site, are used in the design of newdrugs.

A conventional application of theoretical techniques to the process ofdesigning a new drug would be to take a particular protein, for examplean enzyme, and to look at the detailed experimental structure of acomplex between the protein and its ligand, for example a substrate orinhibitor. On the basis of the structural details of IL-13 and itsbinding site for GAGs, new compounds are proposed. The compounds arescreened by molecular modeling, using the technique known as dockingcalculations, which explore many mutual orientations of protein andligand to find out how best to accommodate the ligand in the bindingsite. Compounds with the best affinity for the protein producetheoretical complexes with favorably low interaction energies.

Example 3 BIAcore Screening Assay

The optical phenomenon of surface plasmon resonance is used to monitorphysical interactions between molecules. Passing a solution of apotential protein ligand (e.g. IL-13) over a sensor surface to which atarget (e.g. heparin) is coupled monitors the real-time binding ofprotein ligands to the immobilized target. Detection is achieved bymeasuring refractive index changes very close to the sensor surface.When the refractive index is altered, the angle at which plasmonresonance occurs changes and this change directly correlates with theamount of protein interacting with the surface. A BIAcore 2000 isconveniently used. It is very sensitive and its microfluidics ensuresthat only small amounts of material are required.

Biotinylated heparin is immobilized on the biosensor chip. Biotinylationoccurs via amino groups, or reducing termini modified with ammonia byreductive amination, using sulfo-NHS-biotin. Solutions containingpotential protein ligands of interest are injected over the sensor chipsurface, and the binding is measured in real time (Femig, In:Proteoglycan protocols, Ed. R. V. Iozzo, Humana Press, Totowa, N.J.,USA, 2001). Bacterially expressed recombinant human IL-13 (rhIL-13)readily binds to heparin immobilized by this method. Indeed, rhIL-13bound best when the binding buffer was slightly acidic (pH 6), (FIG. 1)and this binding was specific as there is little interaction of rhIL-13with sensor chips lacking heparin. Moreover, the binding of rhIL-13 toheparin is concentration dependent (FIGS. 1A and 1B).

Preparations of the anionic glycoconjugate, pentosan polysulfate (PPS)inhibit the binding of IL-13 to heparin immobilized on the BIAcore chip.Titration experiments indicate that PPS is a better inhibitor of IL-13binding to immobilized heparin than heparin itself. These data indicatethat PPS is binding to the same site on IL-13 that heparin binds.

PPS is a sulfated xylan. Not all sulfated xylose polysaccharides bindIL-13, the size of the sulphated xylose polysaccharides is an importantcomponent of its ability to bind to IL-13 and thereby block IL-13binding to heparin. Small sulfated xylans of degree of polymerization(D.P.) 4 or less were unable to inhibit IL-13 binding to immobilizedheparin.

Example 4 Functional Analyses of Heparin on the Target Protein, IL-13

Heparin inhibited the proliferation of a human IL-13 responsive cellline. This occurs at very low doses and is not due to a toxic effect ofthe heparin because other, similarly sulfated polysaccharides, at thesame concentrations of IL-13 and polysaccharide have no effect. Theseexperiments utilize the TF-1.8 cells. TF-1.8 cells are a subclone of theTF-1 cells that have been selected for growth in IL-4, and because IL-4and IL-13 share a receptor these cells are also responsive to IL-13.TF-1 cells were originally established from a bone marrow sample from amale with severe pancytopenia. These cells are dependent on IL-3 orGM-CSF for long term growth and are responsive to a variety of cytokinesincluding IL-4 and IL-13.

TF-1.8 cells have been transfected with the firefly luciferase genecontained in the expression vector, pPGK-puromycin-luciferase (Coombe etal, J. Immunol. Methods 215:145-150, 1998). The positive transfectantsare cloned to produce a line with good luciferase expression. Theproliferation assays are carried out in 96-well microplates suitable forsuch assays (Falcon). The wells are flat bottomed, with white sides anda clear bottom. Cells are washed to remove any cytokine in the growthmedium and then resuspended in RPMI/5% w/v FCS. The cells are countedwith a Coulter Z2 Particle Counter and Size Analyzer (CoulterElectronics, England) and routinely 2.5×10⁴ cells are added tomicroplate wells that contain either no IL-13 (negative control) orvarious dilutions of IL-13. When the effect of PPS, or other sulfatedpolysaccharides is to be measured, the wells also contain variousconcentrations of these molecules.

The cells proliferate for 48 hours at 37° C. in a humidified atmosphere,after which the luciferase activity is measured by the addition of 50 μlof luciferase substrate buffer (50 mM Tris-HCl, pH 7.8, 15 mM MgSO₄,33.3 mM DTT, 0.1 mM EDTA, 0.5 mM Na-luciferin, 0.5 mM ATP, 0.25 mMlithium Co A and 0.5% v/v Triton X-100). Immediately after the additionof the luciferase buffer the plate is assayed for luciferase activity.Light emissions are detected on a Victor 1420 Multilabel counter(Wallac, Turku, Finland).

Using this assay, the inventors demonstrated that heparin markedlyinhibits the IL-13 dependent proliferation of TF-1.8 cells (FIGS. 2A andB) giving 75-80% inhibition of IL-13 mediated cell proliferation atconcentrations of 5-10 μg/ml and 55-60% inhibition at the lowerconcentrations of 2.5-1.25 μg/ml. Heparin at 10 μg/ml was a veryeffective inhibitor of cell proliferation over a wide range of IL-13concentrations (FIG. 2A). Moreover, sucrose octasulfate at aconcentration of 10 μg/ml has very little inhibitory activity of IL-13mediated cell proliferation even at low IL-13 concentrations (FIG. 2A).

The ability of fluorescent-labeled IL-13 to bind to its receptor, in thepresence or absence of heparin, is examined. These experiments areperformed using different concentrations of IL-13, which has beenconjugated with AlexaFluor-488, and heparin. Comparisons with othersulfated polysaccharides shown not to have activity in inhibiting theIL-13 dependent proliferation of TF-1.8 cells, demonstrates specificity.

Example 5 Heparin and Heparan Sulfate Oligosaccharides Bind IL-13

HLGAGs may be partially digested by a number of means, includingenzymatic digestion with heparinases and chemical digestion using agentssuch as nitrous acid, alkaline β-elimination and oxidation inconjunction with alkaline depolymerization (Conrad, Heparin bindingproteins. Academic Press, San Diego, 1998). The enzymes heparinase I andheparinase III cleave at specific sites on the heparin/heparan sulfatechain: heparinase I at IdoA residues with N-sulfated glcN domains, andheparinase III at GlcA residues in unsulfated N-acetyl GlcN domains.

Heparan sulfates are depolymerized according to the procedures describedby Turnbull et al. (Proc. Natl. Acad. Sci. USA 96(6): 2698-2703, 1999),Heparin was depolymerized in accordance with the procedure described byChai et al. (Anal. Chem. 70(10): 2060-2066, 1998). Briefly, heparin (5g) and albumin (4 mg) were dissolved in 50 ml 30 mM CH₃CO₂Na, containing3 mM CaCl₂ and adjusted to pH 7 with 0.2 M NaHCO₃. Heparinase I, EC4.2.2.7, (2 IU) was added and the mixture incubated at 30° C. for 16hrs. The mixture was boiled for 3 minutes, aliquoted into small volumes(5 ml) and frozen. Aliquots were thawed, centrifuged and filtered beforeinjection (1 ml) on the size-exclusion chromatography system.

SEC was performed on a two 90×1.5 cm glass columns connected in series.The first column was packed with P6 fine and the second with P 10 fine.The columns were eluted with 0.25 M NaCl at a flow rate of 0.25 ml/minusing a Gilson model 307 titanium pump (Middleton, Wis., USA) and theeffluent monitored with a Shimadzu RID-10 refractive index detector(Melbourne, Victoria, Australia). Data was acquired using GilsonUnipoint software. Fractions of 1 ml were collected. Fractions adjacentto the peak maxima were pooled, lyophilized and desalted on a fastdesalting column. The desalted fragments were lyophilized, redissolvedin water and stored at −20° C. The concentration of each fragment wasdetermined spectrophotometrically at 232 nm in 30 mM HCl using theextinction coefficient of 5500 mol⁻¹ cm⁻¹. The size of theoligosaccharides were confirmed using MALDI MS (vide infra).

This library of heparin and heparan sulfate fragments of uniformsaccharide number was then used in the BIAcore assay to determine theirability to inhibit the binding of IL-13 to immobilized heparin. Theseexperiments indicate that pools of heparin fragments of size DP6 or lessare too small for effective binding to IL-13 and thereby blocking IL-13from binding to immobilized heparin. Heparin oligosaccharides DP10 andlarger were effective in this assay to varying degrees, but full lengthheparin or heparan sulfate were the most effective (FIG. 3A).

The library of heparin and heparan fragments of uniform saccharidenumber were also used in a TF-1 cell proliferation assay to determinewhether all saccharides had the same ability to inhibit IL-13 stimulatedcell proliferation. These experiments indicated that not all heparin orheparan sulfate oligosaccharides had the same ability to inhibit IL-13activities. Small heparin fragments (DP6) had little effect whereaslarger heparin fragments (DP10) were more effective (FIG. 3B). Similarlysmall heparan sulfate fragments (DP8) were ineffective inhibitorswhereas larger fragments (DP14) were as effective in this assay as fulllength heparan sulfate (FIG. 3B).

Example 6 Functional Analyses of Pentosan Polysulfate on the TargetProtein, IL-13

PPS inhibited the proliferation of a human IL-13 responsive cell line.This occurs at very low doses and is not due to a toxic effect of thepentosan polysulfate because other, similarly sulfated polysaccharides,at the same concentrations of IL-13 and polysaccharide have no effect.These experiments utilize the TF-1.8 cells. TF-1.8 cells have beentransfected with the firefly luciferase gene contained in the expressionvector, pPGK-puromycin-luciferase (Coombe et al, 1998 supra). Theproliferation assays are carried out in 96-well microplates suitable forsuch assays (Falcon). The wells are flat bottomed, with white sides anda clear bottom. Cells are washed to remove any cytokine in the growthmedium and then resuspended in RPMI/5% w/v FCS. The cells are countedwith a Coulter Z2 Particle Counter and Size Analyzer (CoulterElectronics, England) and routinely 2.5×10⁴ cells are added tomicroplate wells that contain either no IL-13 (negative control) orvarious dilutions of IL-13. When the effect of PPS is to be measured,the wells also contain various concentrations of this molecule.

The cells proliferate for 48 hours at 37° C. in a humidified atmosphere,after which the luciferase activity is measured by the addition of 50 μlof luciferase substrate buffer (50 mM Tris-HCl, pH 7.8, 15 mM MgSO₄,33.3 mM DTT, 0.1 mM EDTA, 0.5 mM Na-luciferin, 0.5 mM ATP, 0.25 mMlithium Co A and 0.5% v/v Triton X-100). Immediately after the additionof the luciferase buffer the plate is assayed for luciferase activity.Light emissions are detected on a Victor 1420 Multilabel counter(Wallac, Turku, Finland).

Using this assay, the inventors demonstrated that PPS markedly inhibitsthe IL-13 dependent proliferation of TF-1.8 cells (FIG. 2B) with 75-80%of IL-13 mediated cell proliferation blocked at PPS concentrations of5-10 μg/ml.

The ability of sulfated xylans to inhibit IL-13 mediated cellproliferation is size dependent. These experiments utilized the TF-1cell line from which the TF1.8 cells were derived. The assay isperformed as described above with the TF1.8 cells the differences beingthe concentration of IL-13 required for cell growth and the fact thatcell number is determined using a dye. Briefly, proliferation assays arecarried out in 96-well microplates suitable for such assays. Cells arewashed to remove any cytokine in the growth medium and then resuspendedin RPMI/5% w/v FCS and routinely 2.5×10⁴ cells are added to microplatewells that contain either no IL-13 (negative control) or variousdilutions of IL-13. When the effect of the different sized sulfatedxylans was measured, the wells also contained various concentrations ofthese molecules and the IL-13 concentration was held constant at 2.5ng/ml. The cells proliferated for 48 hours, after which the number ofcells present was quantified by staining with 204 per well of theAQUEOUS ONE dye for 3 hours and then absorbance was read at 490 nm.Smaller polysaccharides comprising 2 or 3 sulfated xylose units 1-4linked were ineffective inhibitors when used at either 2.5 μg/ml or 105μg/ml. The larger polysaccharides comprising 4, 5, 7, or 8 sulfatedxylose units linked 1-4 inhibited IL-13 dependent TF-1 cellproliferation but the most effective were septa- and octa-saccharidesand a mixture of sulfated xylose polysaccharides all larger than anoctasaccharide. (FIG. 4 shows the data for the effect of sulfated-xylansof size DP2, DP4, DP5, DP7 and DP8 on the IL-13 dependent proliferationof TF-1 cells

The ability of fluorescent-labeled IL-13 to bind to its receptor, in thepresence or absence of PPS, is examined. These experiments are performedusing different concentrations of IL-13, which has been conjugated withAlexaFluor-488, and PPS. Comparisons with other sulfated polysaccharidesshown not to have activity in inhibiting the IL-13 dependentproliferation of TF-1.8 cells, demonstrates specificity.

Example 7 Docking Protocol for Identification of Heparin Binding Siteson IL-13 Surfaces

The docking strategy developed identifies potential heparin bindingsites on protein surfaces, for use both in the illustration andrationalization of experimental results such as NMR titrations or thedesign of site-directed mutagenesis experiments. As it is notnecessarily the case that heparin structures will bind to proteins in asingle, defined orientation, no emphasis is placed on the detailedprediction of the geometry of the complexes, or accurate calculation ofthe interaction energy. Rather than the conventional use of dockingtechniques, in which the geometry of a small ligand molecule in a knownhigh-affinity binding site is optimized, protocol is used to screen theentire surface of a small protein for clusters of basic residues whichoffer suitable shape and charge profiles complementary to the pattern ofacidic substituents along the heparin chain.

Docking of several heparin oligosaccharide ligand models to proteinstructures is performed as previously described (Forster and Mulloy,Biochem. Soc. Trans. 34:431, 2006) using Autodock, version 2.4 (Morriset al, J. Comput. Aided Mol. Des. 10:293, 1996), with partial chargesfor protein atoms taken from the AutoDock version of the AMBER forcefield. Co-ordinates for the heparin oligosaccharide ligands were derivedfrom the NMR structure for the predominant repeating disaccharide ofheparin lhpn.pdb (Mulloy et al, Biochem. J. 293:849, 1993) with partialatomic charges from ab initio calculations using the Jaguar program(Schrodinger Inc, Portland, Oreg., USA) on 1-OMe 4-OMe substitutedmonosaccharides. Two pentasaccharide ligands were used, each withglucosamine at both reducing and nonreducing termini. For one of these,the two iduronate residues were both in the ¹C₄ conformation, and forthe other they were both in the ²S₀ conformation; in heparin both theseforms are in equilibrium. All the exocyclic bonds in thesepentasaccharide models were regarded as rotatable with the exception ofthe glycosidic linkages. Some calculations were also performed using acompletely rigid endecasaccharide ligand model.

Docking is typically performed on a grid of 120×120×120 points, with theaddition of a central grid point. The grid was centred on the mean ofthe coordinates of the protein. Grid spacing was 0.7 Angstroms, leadingto a grid of 84×84×84 Angstroms. This determines the largest proteinthat can be studied by this protocol. Separate grids of VDW interactionenergy are then calculated for each atom type in the ligand (C, N, H, S,O) and an electrostatic interaction energy grid is computed for a singleelectron charge. These grids are used during the docking process torapidly calculate the interaction energy of the ligand with the protein.This is achieved by finding the grid points surrounding each ligand atomand using an interpolation procedure to find the energy contribution atthe current coordinates. The energies are then summed over all atoms inthe ligand and the torsion energy terms added to the VDW andelectrostatic energies. During the docking procedure the position,orientation and allowed torsion angles of the ligand structure areoptimized by a monte carlo simulated annealing procedure. Initialsimulation temperature (defined in RT units) of 1000 was used and atemperature reduction factor of 0.95 per cycle was used; typically 128runs of 300 cycles were performed.

These parameters were selected in a validation study of the protocol, byperforming simulations on a protein/heparin oligosaccharide complex ofknown crystal structure, that of FGF2 with a heparin hexasaccharide(lbfc.pdb) [Mulloy and Forster, Glycobiology 10:1147, 2000], adjustingparameters to most reliably reproduce the known heparin binding site.Docking is performed with a unit dielectric rather than a distantdependent dielectric as this was found more reliable in reproducingknown binding sites. Docking calculations typically requiredapproximately 50 minutes on a 300 MHz SGI octane workstation. Dockedligand coordinates are extracted from output files using a set ofin-house PERL scripts.

Example 8 Use of Docking Calculations to Screen the Structure Databasefor Heparin-Binding Proteins

The docking protocol is readily be performed on a medium-high throughputbasis, so that the possibility arises of a systematic survey of proteinswith known three-dimensional structures, in order to supplement thelimited number of experimentally determined heparin-protein complexes(Imberty et al, Carbohydr. Res. 342:430, 2007). There are over 41,000structures in the PDB, however, so an initial survey of a subset ofsolved protein structures is desirable. In the SCOP (StructuralClassification of Proteins) [Murzin et al, J. Mol. Biol. 247:536, 1995]system (http://scop.mrclmb.cam.ac.uk/scop/), members of the Superfamilyof 4-helical cytokines (in the Class of all-alpha proteins) includingIL-13 form a suitable group. They are small proteins, related infunctional terms as well as by structure, and all performing theirbiological functions outside the cell, so that their environment is richin glycosaminoglycans.

Semi-automated docking calculations are performed using the programAutodock, as previously described (Forster and Mulloy 2006, supra), withco-ordinates for the heparin-based oligosaccharide ligands taken fromthe PDB file lhpn.pdb (Mulloy et al 1993, supra) and co-ordinates forhuman IL-13 derived from PDB files (http://www.rcsb.org/pdb/) PDB ID:1ijz. Only the structure of human IL-13 cytokines were chosen.Heparin-protein complexes calculated to have intermolecular interactionenergies of less than −1000 kcal/mol are regarded as predictions ofcapacity to bind heparin; those with interaction energies of more than 0were regarded as predicting no capacity to bind heparin. The energyunits of the Autodock function are usually given as kcal/mol; the highfigures in the Tables are a consequence of the high weighting given toelectrostatic terms in the forcefield by using a unit dielectricconstant. Values given should be understood as the results of a rankingfunction with no significance in absolute terms. Co-ordinates arevisualized and figures prepared using the programs Insight ll and WeblabViewer (Accelrys).

The binding properties of heparin pentasaccharides interacting withhuman IL-13 are listed in Table 3.

TABLE 3 Prediction of heparin binding to IL-13 IntermolecularInteraction Energies for Position Cytokine PDB pentasaccharide ligandsof heparin (abbreviation) filename IdoA¹C₄ IdoA²So binding site²Interleukin-13 lijz.pdb −1099 −1120 AB loop (K25) (monomer) and Helix D(K97, K104, K105, R108)

Docking was also performed using a completely rigid heparinendecasaccharide and the results of this analysis are illustrated inFIG. 5.

Example 9 Determination of the Heparin Binding Site on IL-13

FIG. 1 shows the BIAcore binding curves of wild-type IL-13 binding toheparin immobilized on a biosensor chip. A number of differentconcentrations of IL-13 are shown.

Site directed mutagenesis was performed on IL-13. Basic residues andsome acidic residues within the proposed heparin binding site werechanged to alanine (A) and the proteins expressed using the baculovirusexpression system. Insect cell expressed proteins were purified oneither a monoclonal anti IL-13 antibody affinity column or a polyclonalanti-IL-13 antibody affinity column and checked for purity by SDS-PAGEand silver staining. Mutant IL-13 proteins were examined for theirability to bind heparin immobilized on a biosensor chip and binding wasassessed using a BIAcore 2000. Binding curves for wild type IL-13 andsome of the mutant proteins are shown in FIG. 6. These data indicatethat amino acid residue numbers K25 in the AB loop and K97, H102, K104,K105, R108, and R111 are key residues in the heparin binding site onwild type IL-13. The orientation of these critical amino acids in IL-13for heparin binding are shown in FIG. 5. More particularly these dataindicate that heparin binds to both of the common forms of IL-13, thewild type where arginine is in the 111 position and the polymorphic formwhere glutamine is in position 111 (IL-13Q111), although wild type IL-13binds heparin more strongly. From the molecular modeling it appears thatR111 does not directly interact with the heparin chain but it doescontribute to the overall basic charge of the C-terminal region of the Dhelix and because of this heparin binds the R111 form of IL-13 moreeffectively (FIG. 5 and FIG. 6).

To obtain an indication as to the ability of heparin to bind the variousIL-13 mutants in solution the ability of soluble heparin to bind thevarious IL-13 mutants and so block the interaction of the mutants withimmobilized heparin was determined. Heparin was immobilized on a BIAcorebiosensor chip surface and the IL-13 mutants plus various concentrationsof soluble heparin were in the fluid phase. The concentration of heparinrequired to inhibit the IL-13 proteins binding by 50% (IC₅₀) weredetermined and these data are shown in Table 4.

TABLE 4 Ability of soluble heparin to inhibit IL-13 proteins frombinding to immobilized heparin IC₅₀ nM IL-13 protein End point heparin*Low density heparin* IL-13 WT (R111) 25 3 IL-13Q111 K25A 50 4.5IL-13Q111 K97A 50 8 IL-13Q111 H102A 30 4.5 IL-13Q111 K104A 50 8 *Twodifferently prepared biosensor surfaces were used. The end point heparinsurface was coupled with heparin biotinylated at the reducing terminus.The low density surface was prepared with heparin biotinylated along theGAG chain. These biotinylation methods and methods of coupling heparinto biosensor surfaces are described in Osmond et al, Analyt. Biochem.310: 199-207, 2002.

Molecular modeling calculations of the interaction energies are ingeneral accordance with the experimental data. These calculations havebeen performed for mutations on both backgrounds of IL-13: the Q111 formand the R111 form, and heparin fragments comprising 11 saccharides and 5saccharides have been modeled. In addition the heparin pentamer has beenmodeled in the ¹C₄ chair and the ²S₀ skew-boat conformation of theiduronic acids and the free energies of interaction have been calculatedfor each. These data are given in Table 5 and Table 6. In all cases theinteraction is stronger for the longer heparin fragment compared to theheparin pentamer regardless of the iduronic conformation.

TABLE 5 Interaction energies of the docked heparin oligosaccharidesbinding to human IL-13: IL-13Q111 background* IL-13 protein* Heparin11-mer 5-mer ¹C₄ 5-mer ²S₀ 1IJZ −1734 −1099 −1120 IL-13Q111 −1441 −889−912 K104A −861 −613 −572 K105A −947 −625 −608 K25A −1038 −810 −810R108A −882 −595 −606 H102A −1441 −927 −923 K97A −923 −716 −728 *Startingco-ordinates: 1IJZ.pdb, IL-13 monomer, 111R variant 111q variant andlisted mutants built by residue replacement in Insight, no furtherminimization as all the basic residues were fully exposed, exceptpossibly for H102

TABLE 6 Interaction energies of the docked heparin oligosaccharidesbinding to human IL-13: IL-13R111 background* IL-13 protein* Heparin11-mer 5-mer ¹C₄ 5-mer ²S₀ 1IJZ −1734 −1099 −1120 K104A −1121 −701 −706K105A −1207 −759 −756 K25A −1386 −998 −1014 R108A −1184 −694 −685 H102A−1729 −1098 −1125 K97A −1273 −932 −928 *Starting co-ordinates: 1IJZ.pdb,IL-13 monomer, 111R variant Listed mutants built by residue replacementin Insight, no further minimization as all the basic residues were fullyexposed, except possibly for H102

Example 10 The Site of Heparin Binding to the IL-13 Proteins Overlapswith the Receptor Binding Sites

It was demonstrated in FIG. 2 and FIG. 4 that heparin could inhibit theproliferation of TF1.8 cells and TF-1 cells. A key part of the mainheparin binding site on IL-13 is on helix D and is centred around thebasic amino acids K97, H102, K014, K105 and R108. Amino acids in theD-helix have also been described as important for binding to IL-13Rα1and/or IL-13Rα2, these are 11102, K104, K105, R108, E109 and R111 (Arimaet al, 2005 supra; Madhankumar et al, 2002 supra). These data aresupported by molecular modeling studies and the crystal structures ofthe signaling complex of IL-4Rα/IL-13/IL-13Rα1. Analyses of the crystalstructure further suggest K104 and R108 on IL-13 are criticallyimportant for the interactions with IL-13Rα1 domain 3 (LaPorte et al,2008 supra). A stripe of amino acids on the A and D helices demarcates ahydrophobic canyon lined by the alkyl moieties of these amino acids.These side chains form clefts into which the receptors insert to contactthe main chains of the cytokine A and D helices and of particularimportance are the side chains of amino acids R108 and K104 on the Dhelix. Whereas it appears that R111 is important for binding to thesoluble receptor IL-13Rα2 (Andrews et al, 2007 supra). Domain 1 ofIL-13Rα1 interacts with a hydrophobic saucer-shaped patch formed by thealkyl side chains of M33, D87, K89, T35 (LaPorte et al, 2008 supra).

The concentration of each of the IL-13 mutants required to produce 50%of the maximal TF-1 cell proliferation was determined and these data aregiven in Table 7. The IL-13R111 wild type and the IL-13Q111 variant areshown for comparison.

TABLE 7 Concentration of IL-13 required for 50% TF-1 cell proliferationED50 ng/ml IL-13 proteins ave stdev R111 wt 1.2 0.58 Q111 1.26 0.51(Q)H102A 5.31 0.83 (Q)K104A 46.22 12.2 (Q)K105A 22.63 2.4 (Q)R108A 3.20.65 (Q)K97A 1.33 0.38 (Q) K25A 2.64 0.48

The data in Table 7 agree with that published indicating the prime roleof amino acids on the C-terminal end of the D helix for IL-13 function.

The cell proliferative activity of both the main polymorphic forms ofIL-13: IL-13Q111 and IL-13R111 are comparable in the TF-1 cell assay andboth are equally sensitive to inhibition by heparin FIGS. 7A and B. TheTF-1 cell proliferation assay was used to obtain these data. Details ofthe assay are given in Example 6. This clearly indicates that heparin ora heparin fragment or a polyanionic polysaccharide, or a polyanionicglycoconjugate can effectively inhibit the biological activity of boththe naturally occurring forms of IL-13.

Molecular modeling of heparin fragments binding to IL-13Q111 andIL-13R111 reveals that the heparin fragments bind to IL-13 at a sitethat overlaps that recognized by domain 3 of IL-13Rα1. The heparinchains bind to both IL-13Q111 and IL-13R111 with approximately the sameorientation (FIG. 8). This means that the mechanism for inhibiting thebiological activity of the two forms of IL-13 are identical.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

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What is claimed is:
 1. A method of identifying an agent as effective fortreatment of an inflammatory process in a subject, said methodcomprising: determining whether said agent interacts with a polyanionicglycoconjugate-binding site on IL-13, and identifying said agent aseffective for said treatment if it interacts with said binding site. 2.The method of claim 1, wherein the polyanionic glycoconjugate-bindingsite on IL-13 selected from the group consisting of an amino acid in theAB loop and an amino acid in helix D of human IL-13 or the equivalent ina non-human IL-13.
 3. The method of claim 1 wherein the polyanionicglycoconjugate is a glycosaminoglycan (GAG).
 4. The method of claim 3wherein the polyanionic glycoconjugate-binding site is selected from thegroup consisting of: (i) a conformation of amino acid residuescomprising Q22, Q24 and K25 in the AB loop of IL-13 or its equivalent ina non-human IL-13; (ii) a conformation of amino acid residues comprisingK97, D98, H102, K104, K105, R108, E109 and R111 in helix D of humanIL-13 or its equivalent in non-human IL13; and (iii) a conformation ofamino acid residues comprising Q22, Q24 and K25 in the AB loop and K97;D98, H102, K104, K195, R108, E109 and R111 in helix D of IL-13 or itsequivalent in non-human IL-13 or its equivalent in non-human IL-13. 5.The method of claim 4 wherein the polyanionic glycoconjugate-bindingsite is Q22, Q24 and K25 of the AB loop and K97, D98, H102, K104, K105,R108, E109 and R111 of human IL-13 or its equivalent in non-human IL-13.6. The method of claim 1 wherein the agent is selected from the groupconsisting of a GAG, heparin and pentosan polysulfate (PPS) and afraction or derivative thereof.
 7. The method of claim 1 wherein theinflammatory process is selected from the group consisting ofinflammation, fibrosis, chronic graft rejection,-stem celldifferentiation and stem cell proliferation.
 8. The method of claim 7wherein the inflammation is allergic inflammatory disease.
 9. The methodof claim 8 wherein the allergic inflammatory disease is selected fromthe group consisting of asthma, allergic rhinitis, chronic obstructivepulmonary disease (COPD) and acute respiratory distress syndrome (ARDS).